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Main Content
Quantifying functional biodiversity [electronic resource] / Laura Pla, Fernando Casanoves, Julio Di Rienzo.
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Record details
- Author/Creator:
- Pla, Laura E.
- Publication:
- Dordrecht ; New York : Springer, c2012.
- Series:
SpringerBriefs in environmental science.
SpringerBriefs in environmental science - Format/Description:
- Book
1 online resource (ix, 98 p.) : ill. - Subjects:
Biodiversity -- Statistics. - Notes:
- Description based on online resource; title from digital title page (viewed on Feb. 29, 2012).
Includes bibliographical references and index.
Electronic reproduction. Palo Alto, Calif. Available via World Wide Web. - Local notes:
- Acquired for the Penn Libraries with assistance from the Dr. D. Sergeant Pepper Memorial Fund.
- Contributor:
Casanoves, Fernando.
Di Rienzo, Julio.
ebrary, Inc.
Dr. D. Sergeant Pepper Memorial Fund. - ISBN:
- 9789400726482 (electronic bk.)
9400726481 (electronic bk.) - OCLC:
- Publisher no.:
- 99947290120
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Page 1
Experimental library screening demonstrates the
successful application of computational protein
design to large structural ensembles
Benjamin D. Allena,1, Alex Nisthalb,1, and Stephen L. Mayoc,2
aDivision of Chemistry and Chemical Engineering,
Engineering, California Institute of Technology, MC 114-96, 1200 East California Boulevard, Pasadena, CA 91125
bBiochemistry and Molecular Biophysics Option, and
cDivisions of Biology, and Chemistry and Chemical
Contributed by Stephen L. Mayo, September 7, 2010 (sent for review April 20, 2010)
The stability, activity, and solubility of a protein sequence are
determined by a delicate balance of molecular interactions in a
variety of conformational states. Even so, most computational
protein design methods model sequences in the context of a single
native conformation. Simulations that model the native state as an
ensemble have been mostly neglected due to the lack of suffi-
ciently powerful optimization algorithms for multistate design.
Here, we have applied our multistate design algorithm to study
the potential utility of various forms of input structural data for
design. To facilitate a more thorough analysis, we developed
new methods for the design and high-throughput stability deter-
mination of combinatorial mutation libraries based on protein
design calculations. The application of these methods to the core
design of a small model system produced many variants with
improved thermodynamic stability and showed that multistate
design methods can be readily applied to large structural ensem-
bles. We found that exhaustive screening of our designed libraries
helped to clarify several sources of simulation error that would
have otherwise been difficult to ascertain. Interestingly, the lack
of correlation between our simulated and experimentally mea-
sured stability values shows clearly that a design procedure need
not reproduce experimental data exactly to achieve success. This
surprising result suggests potentially fruitful directions for the
improvement of computational protein design technology.
protein engineering ∣ high-throughput stability determination ∣ library
design ∣ molecular dynamics ∣ NMR ensemble
based computational protein design (CPD) methods have been
developed to allow screening for desirable sequences to be
performed in silico (4–6). Despite a number ofhigh-profile results
that demonstrate the utility of CPD (7–12), the routine computa-
tional design of functional proteins remains elusive. Thus, many
current efforts focus on the improvement of CPD methodology
or on the synergistic application of CPD with experimental
high-throughput screening or selection (13).
Although the stability, solubility, and activity of a protein
depend on the relative energetic contributions of many confor-
mational states, including ensembles of native, unfolded, and ag-
gregated structures (14), most CPD methods evaluate sequences
based on their energies in the context of one fixed-backbone
structure. This simplification has made design results undesirably
sensitive to slight changes in main-chain and side-chain confor-
mation and has made difficult the selection of sequences with
amino acid composition similar to naturally occurring proteins.
These issues have been approached via the use of high-resolution
structural templates, expanded rotamer libraries (15, 16), energy
functions with softened repulsive terms (10, 17, 18), iteration
between structural refinement and sequence design (10, 19),
and amino acid reference energies (10, 20). Although these stra-
tegies can help to mitigate the impact of the fixed-backbone
rotein-engineering efforts based on directed evolution have
met with considerable success (1–3). In tandem, structure-
approximation, they do not address the fundamental reality that
sequence fitness is a function of multiple conformational states.
In a handful of cases, multistate design (MSD) procedures
have been used to find sequences that simultaneously stabilize
or destabilize a combination of a few different conformational
states (21–23). However, MSD techniques have not yet been
applied to native ensembles with many conformational states that
might better reflect the flexibility of real proteins. The degree to
which various energy functions, rotamer libraries, and structural
templates of single-state design (SSD) might be appropriate for
this type of MSD calculation is, so far, unknown. We recently de-
veloped a framework for MSD that allows for efficient sequence
optimization given hundreds of conformational states (24). Here,
we have applied this framework to test the applicability of current
CPD methods to large structural ensembles, and to investigate
whether the use of such ensembles might result in the selection
of more desirable sequences by CPD.
The most basic goal of CPD has been to optimize interactions
between amino acid side chains to promote thermodynamic sta-
bility of the native state. Unfortunately, standard methods for the
measurement of protein stability are too laborious to allow the
testing of more than a few designed variants, and the top-scoring
sequence produced by a new design procedure does not yet suffi-
ciently reflect its general utility. Fortunately, recent progress in
laboratory automation has allowed us to construct an efficient
pipeline for the basic evaluation of new procedures in CPD. In
our scheme, gene libraries are assembled from degenerate oligo-
nucleotides, proteins are expressed and purified in microtiter
plates, and liquid-handling robotics assist in the preparation of
chemical denaturation series in a 96-well format for assay by
tryptophan fluorescence. The integration of these technologies
has allowed us to assess the stability of hundreds of designed pro-
tein variants with minimal experimenter intervention and limited
incremental expense.
Given several design procedures to evaluate and a high-
throughput experimental assay, we needed a general and rigorous
tein library design methods have been developed (25–28), none
reported so far takes directly into account simulation energies,
allows control over library size and possible sets of amino acids,
and eschews heuristics that can introduce bias into the libraries
it produces. So that our experimental results might better reflect
the results of the underlying CPD calculations, we developed a
Author contributions: B.D.A. and A.N. designed research; B.D.A. and A.N. performed
research; B.D.A., A.N., and S.L.M. analyzed data; and B.D.A., A.N., and S.L.M. wrote
the paper.
The authors declare no conflict of interest.
1B.D.A. and A.N. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/
19838–19843 ∣ PNAS ∣ November 16, 2010 ∣ vol. 107 ∣ no. 46www.pnas.org/cgi/doi/10.1073/pnas.1012985107
Page 2
unique library design procedure, called Combinatorial Libraries
Emphasizing And Reflecting Scored Sequences (CLEARSS),
which satisfies these criteria.
We used standard single-state design (SSD) and MSD to rede-
sign the core of the small, stable domain Gβ1 based on several
sources of structural information, including a crystal structure,
an NMR structure, and MD simulations. Our efforts were moti-
structural data for design and the hypothesis that use of a struc-
tural ensemble might help to correct for design failures observed
in SSD. Because the imperfect nature of CPD limits the conclu-
developed previously undescribed methods for the computational
design and high-throughput experimental stability determination
of combinatorial protein libraries. The results we report here
provide simultaneous experimental validation for (i) the applica-
tion of multistate protein design methods to large conformational
ensembles, (ii) the transformation of arbitrary CPD results into
combinatorial mutation libraries, and (iii) the experimental stabi-
lity determination of these libraries by high-throughput gene
assembly, protein expression, purification, and screening.
Results and Discussion
Designed Libraries. To simplify the validation of our multistate
design methods, we applied them to a previously studied set of
core positions (Fig. S1) in a small model system, protein Gβ1,
and relied on a set of energy functions that previously found
stabilized variants of this sequence (17). We assessed these meth-
ods by performing designs based on each of the following sources
of structural information: a crystal structure (xtal-1), an NMR-
constrained minimized average solution structure (NMR-1), an
NMR ensemble (NMR-60), a constrained MD ensemble (cMD-
128), and an unconstrained MD ensemble (uMD-128). Our algo-
rithm for library design (Fig. 1) was then applied to produce
degenerate oligonucleotide sequences that reflect quantitatively
Given the requirements for purified protein of our stability assay,
we chose to design and screen a 24-member library based on each
structural data source described above.
All five designed libraries comprise relatively conservative sets
of mutations away from the wild-type sequence (Table 1). The
libraries other than uMD-128 share many characteristics in com-
mon. Each of these libraries chose only the wild-type amino acid
at positions A20, A26, F30, and A34. Every member of each of
these four libraries contained the single-mutant Y3F, which
ture. These four libraries all allowed the wild-type amino acid at
every other position, and all contain the most stable Gβ1 core
variant previously characterized, Y3F þ L7I þ V39I (17).
The two NMR libraries were extremely similar to each other:
Both chose the amino acids FILVat position 52 and directed the
remaining diversity to positions 7 and 39. In contrast, xtal-1 and
cMD-128 allowed only the wild-type Phe at position 52 and
instead allocated diversity toward positions 7, 39, and 54. xtal-1
differs from cMD-128 in that it gave up L7F and V39L to allow
L5I. The unconstrained MD ensemble library uMD-128 was the
least conservative, specifying a size reversal of two nearby resi-
dues via mutations L5A and A34F, and diversity at residue 30,
a position untouched in the other libraries.
As shown in Table S1, the designed libraries generally suc-
ceeded in representing the top-scoring sequences from each
design calculation, given the constraints imposed by the genetic
code. The exception was the uMD-128 library, which represented
only three of the best 100 sequences from the original design
calculation. This was caused by an unusual designed sequence list,
in which the best-scoring sequence contained a feature (the
size reversal mentioned above) that was very uncommon in the
remainder of the list.
Experimental Characterization of Designed Libraries. Experimental
screening of the xtal-1 library (Fig. S2A) showed two distinct sets
of variants. The 12 library members with wild-type Leu at position
5 all exhibited stabilities similar to or better than the wild-type
sequence, while the 12 with Ile at position 5 were all significantly
destabilized. Screening of the NMR-based libraries (Fig. S2 B
and C) showed a similar dichotomy. In each case, the six library
members with the wild-type Phe at position 52 exhibited wild-
type-like stability or better. The remaining 18 variants from each
NMR-based library were highly destabilized, and many lacked en-
ough of a pretransition to be fit to the two-state unfolding model.
Evaluation of the MD libraries indicated that all 24 variants
from the constrained library, cMD-128, had stability similar
to the wild-type protein or better (Fig. S2D). In contrast, all 24
variants from the uMD-128 library failed to produce any signifi-
cant change in fluorescence signal across the denaturation series
and likely assume an alternative structure, as discussed below.
Sorting the individual sequence members from every library
except uMD-128 according to experimental stability (Fig. 2)
shows that the cMD-128 input structural ensemble favored more
high-quality sequences (better than wild type) than any other
library. Every other designed library specified at least one proble-
matic substitution that rendered many of its sequences destabi-
lized or otherwise unlike the wild type.
Impact of Input Structural Data on Designed Libraries. Why were
Fig. 1.
based on computational protein design calculations. A line of boxes indicates
a protein sequence; each box represents a position in the protein chain.
Different colored boxes represent different amino acids. The set of sequences
on the far right corresponds to the expansion of a particular combinatorial
library into the set of sequences it represents. The energies of the sequences
in the expansions are used to decide which combinatorial library to test
experimentally, as described in Materials and Methods.
General scheme used to design combinatorial mutation libraries
Table 1. Combinatorial libraries designed from different sources of
structural information
ResidueWT xtal-1NMR-1NMR-60cMD-128uMD-128
xtal-1: library based on single-state design of the crystal structure. NMR-1:
library based on single-state design of the constrained minimized average
NMR solution structure. NMR-60: library based on multistate design of the
60-member NMR structural ensemble. cMD-128: library based on multistate
design of the constrained molecular dynamics ensemble. uMD-128: library
based on multistate design of the unconstrained molecular dynamics
Allen et al.PNAS
November 16, 2010
vol. 107
no. 46
Page 3
in high-scoring sequences from the original design calculations
rather than artifacts introduced by the library design process.
The selection of the amino acids FILV at position F52 in
the two NMR-based libraries resulted in three quarters of each
library being significantly destabilized. In the context of the NMR
structures, no Phe rotamer in the library was able to fit perfectly
at position 52, encouraging the selection of smaller amino acids.
If the set of rotamers at this position is supplemented with the
observed rotamer in each structure, the designs choose to allocate
diversity to positions 7 and 39, resulting in libraries similar to
xtal-1. This result highlights how dramatically the rotameric ap-
proximation can influence the results of a design, despite our
biophysical intuition that a solution ensemble might better reflect
protein structure than a single crystallographic snapshot. It sug-
gests that, at the very least, rotamers optimized for the wild-type
sequence should be included when the goal is to simply find
desirable sequences. For this project, we omitted the structurally
observed rotamer at each position in order to limit the significant
bias toward the wild-type sequence that these rotamers tend to
cause. In the context of a real-world protein-engineering project,
including these rotamers would have considerably increased our
chances of success. Interestingly, this failure of discrete rotamers,
occurring as it did in the design of the NMR ensemble, indicates
that continuous side-chain optimization may be useful during
design, even when allowing conformational flexibility of the main
The L5I mutation, which caused half of the xtal-1 library
members to be destabilized relative to the wild-type sequence,
may have been selected due to a failure of the softened repulsive
contact potential that is used to counteract unrealistic rigidity
introduced by the CPD model. The γ methyl group of Ile5 bumps
into a Thr residue on an adjacent β strand and is scored as a
serious clash using unscaled van der Waals radii but appears
innocuous with the atomic radius scaling factor of α ¼ 0.9 that
we used for the designs evaluated here (17). Repeating the design
calculations with radii scaled by intermediate values such as 0.925
and 0.95 prevents Ile from being chosen at position 5 but also
increases the frequency with which smaller residues are chosen
at position F52. Interestingly, the recommendation of α ¼ 0.9
is derived from previous experiments based on the same set of
Gβ1 core positions that were designed here. The earlier work
drew conclusions based only on the best-scoring sequences pro-
duced by the design calculations and found no difference between
scaling atomic radii by 0.9 or 0.95 (17). Our results indicate that
the mutations produced by the design procedure varysignificantly
with values of α between 0.9 and 0.95 when more sequences are
taken into account. Therefore, a more rigorous investigation of
appropriate α values for design may be warranted. Although the
L5I mutation might also be reasonably attributed to the fixed
main-chain and discrete rotamers, several good-scoring libraries
based on the constrained MD ensemble also contained this
mutation (see below). Because the additional conformational
diversity provided by the ensemble did not inhibit this design
failure, we find explanations related to energy function more
To analyze the uMD-128 data, it is important to note that our
stability assay reports on the environment of the single Trp resi-
due of Gβ1. Changes in packing caused by substitutions at other
positions could alter the native-state environment of Trp43
enough to flip its side chain out into solution or change its fluor-
escence properties, crippling our ability to monitor unfolding
by fluorescence. This interpretation seems unlikely for the desta-
bilized members of the crystal structure and NMR libraries, for
which a partial unfolding transition is clearly indicated by the
raw data. However, the members of the uMD-128 library fail
to show any such a transition, rendering the validity of our assay
suspect in this case.
A constant feature of the uMD-128 library is a size reversal
specified by mutations A34Fand L5A. The model structures pro-
duced by this design were well packed and contained no obvious
flaws such as Trp43 flipping out into the solvent. Previous char-
acterization of several Gβ1 variants that include mutation A34F
has indicated that these sequences assume oligomeric structures
and exhibit altered fluorescence properties (29–31). This suggests
that the structural basis for our designs, as well as our fluores-
cence assay, may be inappropriate for sequences containing this
mutation. When we reanalyzed a subset of the uMD-128 variants
using circular dichroism, they uniformly displayed wild-type-like
secondary structure but lower stability and low levels of protein
expression. The previous reports and our new results indicate that
the uMD-128 library sequences likely assume structures different
Fig. 2.
the cMD-128, NMR-1, NMR-60, and xtal-1 libraries were named according
to their designed positions (Sequence ID) and sorted by their experimentally
determined Cm value. Some sequences have membership in more than one
library. All sequences above the "wild-type stability" label are more stable
than the wild-type sequence. No sequences below the "unfolded protein"
label gave a measurable transition in the stability assay.
Library mutants sorted by experimental stability. All sequences from
www.pnas.org/cgi/doi/10.1073/pnas.1012985107Allen et al.
Page 4
from the design target. As target structures move away from
experimentally determined structures and greater sequence
diversity is enabled (32, 33), more effective negative design stra-
tegies may be required to exclude sequences that preferentially
adopt alternative conformations.
A recent theoretical analysis of NMR and crystal structures as
templates for design has suggested that some individual members
of NMR ensembles might be more appropriate templates than
others (34). To assess the impact this might have had on our
results, we ranked the members of each structural ensemble by
DREIDING energy (35) and separately by Rosetta energy (36).
We then designed new libraries using only the top 16 energy-
ranked structures from each ensemble using each energy ranking
(Table S2). The two new libraries produced from the NMR struc-
tural ensemble were similar to those from the original design;
both specified diversity at position 52 and contain destabilized
sequences. The library based on the top 16 DREIDING-ranked
sequences from the constrained MD ensemble only specifies
known nondestabilizing substitutions, whereas the top 16 Roset-
ta-ranked structures again gave diversity at position 52. For the
unconstrained MD ensemble, the top 16 Rosetta-ranked struc-
tures gave a library very similar to that produced by the entire
ensemble, and the top 16 DREIDING-ranked structures gave
a library of sequences that appear severely overpacked. In total,
the libraries produced from the top-ranked sequences were simi-
lar to those produced from the full ensembles in four cases and
were worse in the remaining two cases. Based on this post hoc
analysis, our multistate library design procedure seems robust
to the influence of poor templates within each ensemble. How-
ever, more sophisticated methods of template selection may
ultimately prove more fruitful. For example, it might be interest-
ing to choose a subset of a structural ensemble according to the
degree to which individual members are able to recover wild-
type-like sequences and apply MSD to this subset rather than
the entire ensemble.
Influence of the Designed Library Selection Method. At this point, it
is important to address the degree to which the library design
method might affect the conclusions we draw from our experi-
ments. The CLEARSS library design procedure was developed
with an understanding that many different combinatorial libraries
may similarly represent a given list of scored sequences. It is
intended to produce a list of the top-scoring designed combina-
torial libraries that satisfy all constraints and to let the user
choose between them. In general, this choice might be influenced
by chemical intuition or prior mutational data and thus partially
account for properties of the system that are not modeled during
the design procedure. To make our evaluation of input structural
data sources as fair as possible, we chose to ignore such influences
and apply an objective strategy based on the energies of the se-
quences in the libraries. Still, we must ask how the other libraries
generated by CLEARSS would have fared in our experimen-
tal assay.
Each of the top 20 designed libraries based on the NMR en-
semble, and each based on the single average NMR structure,
assigned smaller residues than the wild-type Phe to position
52. The remaining diversity of each library was occupied by var-
ious combinations of the other mutations present in the xtal-1,
NMR-1, and NMR-60 libraries we screened in this work. It seems
very likely, then, that the screening of any of the top NMR-based
libraries from our designs would have resulted in stabilities simi-
lar to those we have reported here. Similarly, all of the top 20
designed libraries based on the unconstrained MD ensemble
contained mutations L5A and A34F and would be expected to
exhibit properties similar to uMD-128.
A more interesting case is provided by the designs based on the
crystal structure and constrained MD ensemble. Our analysis of
the libraries xtal-1 and cMD-128 produced by these designs
seems to indicate that cMD-128 was more successful, because
a much greater fraction of its members were shown to be highly
stable. However, when the top 20 libraries from each design
were inspected in aggregate, it became clear that the xtal-1 and
cMD-128 designs had produced a variety of libraries, some fea-
turing the destabilizing mutations described above. Both the
xtal-1 library and the cMD-128 library were found in the top-20
set of libraries produced by each design. Furthermore, each
design produced several libraries with diversity at position 52, like
NMR-1 and NMR-60.
The influence of the library design procedure on a comparison
between structural inputs can also be assessed by scoring the
sequences from each library on each of the other input structures
or ensembles. Histograms of these energies (Fig. S3) show that
each structural input prefers the sequences from its own library
over those from other libraries, though often by narrow margins.
These observations, taken in total, suggest that the library
design method we used did not unduly influence our optimistic
conclusions about the merits of high-quality structural ensembles
as inputs for computational protein design.
Approximation in Computational Protein Design.In addition to help-
ing validate the use of multistate and combinatorial library design
methods for computational protein design, our results also reflect
unexpectedly on protein design itself. Plots of experimental sta-
bility versus simulation energy for the cMD-128 library (Fig. S4)
failed to yield any correlation, despite the apparent success of this
design calculation. Likewise, the design calculations for xtal-1 and
the NMR libraries failed to predict the pronounced destabilizing
effects of mutations L5I or F52L, even though these designs also
found a variety of stabilized variants.
An intuitive perspective on the development of CPD methods
is that improvements in designed sequences will follow from
improvements in our ability to predict or rank experimental
stabilities (37). However, recent advances in stability prediction
procedures (38, 39) have not yet, to our knowledge, produced the
expected benefits to combinatorial protein design. Our results are
consistent with a recent assessment of stability prediction meth-
ods, which found that the ability to reproduce experimental
stability rankings is unnecessary for useful CPD (40). These con-
clusions prompt a modified view of the factors that make struc-
ture-based design possible in the first place.
Protein structures relax to accommodate mutations, and the
computational difficulty of simulating and scoring these relaxed
structures has so far rendered intractable the accurate stability
ranking of sequence variants with many mutations. Fortunately,
this malleability also means that sequences chosen to fit into a
rigid protein model, even using approximate energy functions,
will likely be tolerated by whatever relaxed structure results from
the mutations they contain. In this way, the soft material proper-
ties of proteins serve to impede the development of the accurate
quantitative protein design methods but also enable the more
qualitative methods we can apply today.
The standard view of CPD has been as a single, rigorously
quantitative problem: Correct packing of amino acid side chains
into a high-resolution template structure leads to a stable and
well-behaved designed sequence. However, our analysis supports
a revised view of CPD, comprising two distinct problems: (i) to
find areas of sequence space that can favorably adopt the target
structure, and (ii) to avoid areas of sequence space that might
favorably adopt alternate structures. The first problem is simply
an enhancement of the original formulation of CPD in which we
admit that current methods for native-state sequence selection
are approximate, and we focus on finding areas of sequence space
enriched with variants that satisfy the target fold.
The second problem has typically been treated implicitly, as
discussed above. The energy function used in this work applies
a simple tripeptide model of the reference state for solvation
Allen et al.PNAS
November 16, 2010
vol. 107
no. 46
Page 5
energies andassumes thatall other interactions average out in the
unfolded ensemble. However, issues such as those encountered
with the uMD-128 library likely cannot be addressed in a general
way without the use of explicit competing state models. Such
simulations are more difficult than those that model only the
native state, in large part because few nonnative states have been
characterized experimentally. In alpha helical peptide systems
where large numbers of undesirable states are readily identifi-
able, explicit negative design has yielded improvements in struc-
tural specificity (41). We hope that general models of unfolded
and aggregated states will lead to similar improvements in the
design of globular proteins.
We enlisted previously undescribed methods for the design and
screening of combinatorial libraries to test the application of
multistate design procedures to several structural ensembles and
to compare the resulting designs to those based on single struc-
tures. Single-state and multistate designs based on NMR data
produced similar sets of libraries; likewise did those based on
crystallographic data. Although an MD-based library gave super-
lative results, we cannot definitively conclude that the use of a
structural ensemble provides any particular advantage over a
single high-resolution structure for the purposes of design. Never-
theless, this initial success confirms that the energy functions and
rotamer libraries developed for single-state modeling are equally
applicable for the multistate design of large structural ensembles.
This work also provides further support in favor of rigorously
screening an area of sequence space discovered by simulation
and has helped in vetting our unique, general method for library
design. For some designs that specified undesired destabilizing
mutations, library screening suggested underlying causes for
design failure that would not have been apparent via the ad hoc
testing of individual sequences. Because our library design pro-
cedure is specifically intended to faithfully represent its input
scored sequence list and is indifferent to the origin of the list,
it should be more useful for the evaluation of new design proce-
dures than its predecessors.
Current design procedures seem to find stable sequences by
selecting mutations that are likely to be accommodated by a
relaxed version of the template structure and not by accurately
ranking the mutations relative to each other. Given that protein
stability and function depend on competing states as well as the
native state, the poor agreement we observed between simulated
and experimental energies in our successful libraries suggests that
future effort toward explicit negative design is warranted.
Materials and Methods
Input Structural Data. Input atomic coordinates for the β1 domain of Strep-
tococcal protein G (Gβ1) were taken from the 2.2 Å crystal structure 1pga
(42), the 60-member NMR structural ensemble 1gb1, and a constrained, mini-
mized average structure generated from the ensemble 2gb1 (43). Hydrogens
(if any) were stripped from each structure, and new hydrogen positions were
optimized along with side-chain amide and imidazolium group flips using
Reduce (44). Each structure was then standardized with 50 steps of conjugate
gradient minimization using the DREIDING force field (35). An unconstrained
128-member molecular dynamics (MD) ensemble was generated from the
minimized crystal structure by running a 12.8 ps MD trajectory at 300 K in
vacuum using the DREIDING force field and saving the coordinates every
0.1 ps. The constrained MD trajectory was generated by the same procedure,
using an additional harmonic point restraint with a force constant of
100 kcal∕mol∕Å2applied to keep Cαatoms near their initial positions. Each
MD snapshot was standardized as described above. After standardization,
the NMR, unconstrained MD and constrained MD ensembles exhibited
average pairwise main-chain rmsds of 0.25, 0.84, and 0.12 Å, respectively.
Sequence Design Specifications and Energy Calculations. In the sequence
designs, 10 core positions of Gβ1 (3, 5, 7, 20, 26, 30, 34, 39, 52, and 54), were
allowed to assume any of the hydrophobic amino acids A, V, L, I, F, Y, and W.
Tryptophan 43 was allowed to change conformation but not amino acid type,
so that our fluorescence-based stability assay would not be compromised.
Allowed side-chain conformations at the variable positions were taken from
the Dunbrack backbone-dependent rotamer library with expansions of ?1
standard deviation around χ1 and χ2 (15). To avoid bias toward the wild-type
sequence, this set was not supplemented with the side-chain coordinates
from the input structure, except at position 43. All other side chains and
the main chain were fixed in the input conformation. Pairwise energies were
computed for each structure or ensemble member using energy functions
described previously (45, 46), with the polar hydrogen burial term omitted.
Sequence Optimization. Fast and accurate side-chain topology and energy re-
finement (FASTER) was used to find optimized sequences in the single-state
design of the crystal structure and the NMR-constrained minimized average
(47). Multistate sequence optimization of each ensemble was performed as
described (24). The energies of a sequence in the context of an ensemble
member were combined into a single score by computing the free energy
of the ensemble system at 300 K:
A ¼ −kT logð∑
where each Ejis the energy of the sequence when threaded on member j of
the ensemble. While various functions could be used to combine the state
energies into a single score, we chose the free energy function over other
averaging schemes because it prefers sequences that satisfy multiple states
in a physically reasonable way that does not require any particular number of
states to be satisfied.
Combinatorial Library Design. To choose combinatorial sequence libraries for
experimental screening, we used a new algorithm reported here (Fig. 1 and
SI Text). Given a list of scored sequences, a list of allowed sets of amino acids,
and a range of desired library sizes, the method evaluates all possible com-
binations of sets of amino acids at different positions that lead to a
library with a size in the desired range. Each position in each library is scored
by summing the Boltzmann weights of the sequences in the list that contain a
library-specified amino acid at that position. The position scores are then
summed to give an overall library score. Our algorithm is able to consider
all possible libraries because it treats positions independently, and because
it ignores amino acid sets that are unnecessarily large in the context of a
given position. In this work, we allowed only those sets of amino acids that
can be specified by degenerate codons that do not include codons observed
with low frequency in Escherichia coli. A temperature of 300 K was used in
the Boltzmann weighting, and the target library size was 24. Setting the
desired library size to other values, such as 12 or 48, gave libraries composed
of the same mutations found in the 24-member libraries.
After applying this algorithm to the lists of sequences produced by the
computational designs, we instantiated the 20 best-scoring libraries from
each design and rescored all of the amino acid sequences in each library
by rotamer optimization. Each library we inspected contained the best-scor-
ing sequence from the design it was based on, although this is not required
by the method. From each design, we chose for experimental testing the
library in the top 20 with the smallest energy spread between its best-scoring
and worst-scoring sequence.
Library Construction, Expression, and Purification. Oligonucleotides (Inte-
grated DNA Technologies) containing approximately 18 bp overlapping
segments were assembled via a modified Stemmer method (48) using KOD
Hot Start Polymerase (Novagen) to generate full-length streptococcal Gβ1
with an N-terminal His6tag. Secondary structure content and annealing
temperatures were verified by nucleic acid package (NUPACK) (49, 50). For
each library, oligonucleotides containing the desired single mutation or
degenerate codon were swapped into the assembly mixture. Standard sub-
cloning techniques were performed to first insert the library into the frame-
shift selection plasmid pInSALect (51) and finally into an expression plasmid
(pET11a). The library was transformed into BL21 Gold DE3 cells (Stratagene),
and colonies were picked into 96-well plates for plasmid miniprepping and
sequencing (Beckman Genomics). Any missing library members were gener-
ated by standard quick-change protocols. Sequence-verified library members
were pulled from replicated glycerol stocks and inoculated into Instant TB
media (Novagen) in 24-well plates. After overnight incubation at 37 °C, cells
were pelleted by centrifugation. Pellets were freeze/thawed once and resus-
pended in 1× CelLytic B (Sigma-Aldrich) lysis buffer before another identical
centrifugation step. Cell lysates were loaded onto an equilibrated HIS-Select
filter plate (Sigma-Aldrich), washed twice, and eluted with buffer containing
250 mM imidazole, pH 8.
www.pnas.org/cgi/doi/10.1073/pnas.1012985107 Allen et al.


text:
No results found.
No results found.
No results found.
Katey Sagal |
Cate Egan Hennessy |
David Spade |
C.J. Barnes (Episodes 40-76; special guest before that, 2004-05) |
Martin Spanjers |
Rory Joseph Hennessy |
James Garner |
Grandpa Jim Egan (Episodes 40-76; special guest before that, 2004-05) |
Kaley Cuoco |
Bridget Hennessy |
Amy Davidson (I) |
Kerry Hennessy |
Raquel Welch |
Jackie |
Guest Star |
Jamie Nations |
director |
Guest Star |
Jill Walter |
receptionist |
Guest Star |
Cate: That agency took a beautiful, confident girl and turned her into a hysterical, dysfunctional mess!
Kerry: Throw in an eating disorder and she could be a model.
Jackie: I'm really good at these things! You're in sixth grade!
Rory: High school freshman.
C.J.: I'd throw it all away to do it again
Kerry: Throw what away? Your room in the basement or your box of dirty magazines?
Grandpa Jim: You want to end up like that crazy nut-bucket?
Bridget: More then anything!
Grandpa Jim: Of course you remember C.J.
Jackie: How could I forget? My receptionist is bringing him up on charges.
Cate: Are you two going to get back together?
Grandpa Jim: I don't see why not. I forgave the Koreans, I can forgive her.
Cate: Do you forgive mom?
Grandpa Jim: No.
Cate: You're not being very nice about mom.
Grandpa Jim: Oh right, the person who left me for Paco the Latin lover.
C.J.: Apparently grandpa and the head of the modeling agency had a fling back in the day.
Cate: Really?
C.J.: I know, gross right?
Grandpa Jim: I'm going down to that modeling agency to give them a piece of my mind.
C.J.: But you have so few left.
C.J.: You pretty thing, why are you cramped behind this desk when you should be out here with the rest of the models?
Receptionist: It's men like you that make me want to date women.
Rory: Where's your career now, big shot?
C.J.: In the toilet with the little sailor guy.
Cate: I don't like the idea of you sprawled out all over the hood of a car.
Rory: Better then in the backseat.
C.J.: Dude that's exactly what I was thinking. Nice! (Cate looks at him) But I had the good mind not to say it.
CJ: Where is the flare, the attitude, the passion...
Kerry: ...the intelligence, the pride, the self-respect?
CJ (to Bridget): Its a dirty, filthy, cut throat buisness. One minute you're pretty, great hair. The next minute you suck, split ends.
Bridget: You guys are never ever in a million years are gonna believe what happened to me at the mall today!
CJ: You flashed a thong to the guy in the food court and he gave you a free soda pop?!
Bridget And.... a total stranger came up to me and asked me to be in a commercial! (giggling) Oh its so good to be me!
Grandpa: I'm gonna go down to that ****ing agency and give them a peace of my mind. And if that make's me old-fashioned, I don't give a horse's patoot!
Rory: No. Saying "horse's patoot" makes you old-fashioned.
Grandpa: Well, how's this, sonny boy? I don't give a rat's a**!
Rory: That's good. But lose the "sonny boy."
6.71 million viewers.
Tape Date: October 22, 2004.
Title: Vanity Unfair
The title is in reference to the magazine Vanity Fair.
User Score: 559
User Score: 529
User Score: 482
User Score: 146
User Score: 120
User Score: 113
User Score: 92
User Score: 70
User Score: 64
User Score: 64
User Score: 63
User Score: 48
User Score: 41
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User Score: 24
User Score: 21


text:
IU to Host Wake Forest Saturday
Feb. 26, 2009 BLOOMINGTON, Ind. - The Indiana University women's tennis team (7-4) host the Demon Deacons of Wake Forest (8-1) on Saturday, Feb. 28 at 11 a.m. inside the IU Tennis Center. Live match updates will be available at IUHoosiers.com. Indiana can take a lead in the all-time series between the schools with a win, breaking the 3-3 tie heading into the contest. The Demon Deacons have won the past two meetings. The Hoosiers look to cool off red-hot Wake Forest as the Decons are coming off a pair of victories over top-25 squads. Wake Forest defeated #25 Texas A&M and #24 William and Mary on back-to-back days last weekend. Also, Wake's Sasha Kulikova is slotted No. 59 in the latest national singles rankings. Indiana opened their 2007-08 season at the Deacon Classic and captured five flight titles, including three singles and both doubles flights. HERBERT CUP WINNERS ONCE AGAIN SOPEL MAKES START WORTHWHILE
Check back soon


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philleykevin's Profile
Title | Last Reply |
Very good recipe. I've probably eaten my weight in oats (enjoyed every bite of it) since I first came across it. Toasted coconut (shaved not shredded) or roasted pecans really sets it off. |
If you want a crunchy exterior you can dust the buttered skillet with cornmeal and bake for a few minutes before adding the batter. It's crunchy but not dry this way. That's the way my family and my wife's family does it. |


text:
Composite parts maker CPI Binani Inc. in Winona, Minn., has invested in a robotic cell container with two Fanuc M-710iC/70 robots. The robots will do repetitive part finishing tasks and do the heavy lifting needed to manipulate parts
Our annual report provides a thorough review and analysis of economic and political conditions, emerging market trends, packaging design advances, materials pricing and sustainable packaging issues impacting supply and demand in the TP segment. Our analysts identify key drivers of growth and opportunities for processors to remain competitive in the years ahead through innovation, production efficiencies and more.
Learn moreThis report is a review and analysis of the North American Plastics Recycling Industry, including key trends and statistics based on 2013 performance. We examine market environment factors, regulatory issues, industry challenges, key drivers and emerging trends in post-consumer and post-industrial recycling.
Learn moreThe annual recap of top trends and future outlook for the plastics caps & closures market features interviews with industry thought leaders and Bill Wood�s economic forecast of trends in growing end markets. You will also gain insight on trends in caps design, materials, machinery, molds & tooling and reviews of mergers & acquisitions.
Learn moreJune 2, 2015 - June 3, 2015Plastics Financial Summit - Chicago 2015
September 15, 2015 - September 17, 2015Plastics Caps & Closures - September 2015
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text:
Pronunciation: /rɛˈljeɪnəʊ/
Definition of relleno in English:
noun ( plural rellenos)
Short for chile relleno.
Example sentences
- When the rellenos were gone Wendy brought out a small chef salad for each of them.
Definition of relleno in:
- US English dictionary
- Spanish dictionary
What do you find interesting about this word or phrase?
Comments that don't adhere to our Community Guidelines may be moderated or removed.
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= trending


text:
Thomet Stables is a family owned and operated boarding and training facility open to all breeds of horses and all disciplines of riding. Modern facilities and compassionate care are our main objectives. We cater to our boarders by offering them personal care, convenient location, affordable pricing and one of the largest facilities in the Grand Rapids area.
Talented, dedicated and caring, the professionals at Thomet Stables offer riding instruction and horse training in a variety of disciplines. Together they are devoted to maintaining a safe, positive learning environment in which students and horses of all ages and levels can thrive.
1900 Pettis Avenue NE, Ada, MI 49301 616-676-3113 email@example.com
Copyright © Thomet Stables, Inc. | All Rights Reserved


text:
Dr. Douglas C. Hingsbergen, MD
- 25 Office Park Dr
Hamilton, OH 45013 - 3 Locations
- (513) 844-1000
Dr. Douglas C. Hingsbergen graduated from the Ohio State University College of Medicine in 1984. He works in Hamilton, OH and 2 other locations and specializes in Surgery. Dr. Hingsbergen is affiliated with Fort Hamilton Hospital, Mercy Health-Fairfield Hospital and TriHealth Evendale Hospital. He speaks English and Spanish.
Gender: Male
Languages Spoken: English, Spanish
Medical School: Ohio State University College of Medicine; Graduated 1984
*This physician has multiple office locations. See Office Information for details.


text:
A Dog Protesting [ 13 photos ] Here is a dog living at central Athens, what makes it special is it's political consiousness.
Facebook, a bastard and a dead dog Read how a jerk got caught running over a poor dog and driving away.
Missing kitten found in fat woman AMANDA Simmons, aka Mandy Mountain says: "My fella wen down on me and found next doors missing kitten"


text:
Hawaiian Dreams: How Joe & Anna Came Together Posted January 7, 2012 by Dan Flaherty Anna, a 28-year-old from Hawaii, had been on CatholicMatch for several months, and while she'd been on some dates, "the one" hadn't yet revealed himself. She was getting… Read More 7 0
From Venice To Jackson Hole: Singles' Summer Travels Posted July 7, 2011 by Christina Ries CatholicMatch members are departing for some fabulous cross-country (and continent) vacations this summer. Here's a sampling. "The Bristol Renaissance Faire in Wisconsin. I went last year with… Read More 5 0


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Keyboard player wanted. Max Middleton or Jan Hammer required for an informal Fusion jam
Do any keyboard players out there want to come and have a Jam over the coming few weeks/months at the Vatican Studios in East London. We consist of a professional drummer an experienced bassist and guitarist that are putting together a set of the f


text:
James Swarbrick
July 1, 2013
by CRC Press
- 4296 Pages
was $3,149.00
Pharmaceutical science deals with the whole spectrum of drug development from start to finish. There are many different facets to the pharmaceutical industry, from initial research to the finished product, including the equipment used, trials performed, and regulations that must be followed. Presenting an overview of all of these different aspects, the Encyclopedia of Pharmaceutical Science and Technology, Fourth Edition is a must-have reference guide for all laboratories and libraries in the pharmaceutical field. Bringing together leaders from every specialty related to pharmaceutical science and technology, this is the single-source reference at the forefront of pharmaceutical R&D.
The strength of this work is not only its breadth but also the caliber of contributing writers, all experts in their field, writing on all aspects of pharmaceutical science and technology. The fourth edition offers 29 new chapters ranging from biomarkers, computational chemistry, and contamination control to high-throughput screening, orally disintegrating tablets, and quality by design. The encyclopedia details best practices of equipment used, methods for manufacturing, options for packaging, and routes for drug delivery. The volumes also provide a thorough understanding of the choices behind each method. In addition, the regulations, safety aspects, patent guidance, and methods of analysis are presented.
Key Areas Covered:
This is an authoritative reference source for those practicing in any area of pharmaceutical science and technology, enabling the pharmaceutical specialist and novice alike to keep abreast of developments in this constantly evolving and highly competitive field.
* Online version coming soon.
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text:
SIMON LAMBERT: Buying a home is the best investment most of us ever make, but ask yourself these questions first
Buying a home is likely to be the best long-term investment most people ever make.
History shows property will probably underperform the stock market over 25 years, but the beauty of a repayment mortgage is that it provides the kind of discipline most investors just don't have - and at the end you'll own an asset worth a small fortune.
Of course, a home is not as flexible as an investment fund - and the cost of running it is higher - but then you can't live in a fund as you build up your holding over a few decades.
Home protection: A house is a great long-term investment but how can you guard against short-term wobbles?
And as you will thus need to rent somewhere to live, so your monthly investment will be limited to the saving between mortgage payments and your rent (if one exists) – and once the mortgage is paid off that difference suddenly swings the other way.
It doesn't deliver the income you can get from dividends, has no earnings to grow and can feel like a heavy burden sometimes, but a home is a long-term appreciating asset that someone will lend you a lot of money to buy at a generally very agreeable rate of interest.
There is also a handy trick whereby wage inflation should dramatically erode your debt over 25 years, by comparison with how rents will typically rise with price inflation over that same period.
If you can put down a reasonable deposit and ride out any storms in your personal finances, then buying home looks like a solid lifetime investment.
The problem is that over the short-term we can get hobbled by the temptation to overpay – and that makes times like now, when house prices are high and mortgage rates are low, particularly risky.
I sit firmly in the camp that believes house prices are too high.
Experian in partnership with This is Money is giving first-time buyers the chance to win £20,000 to put towards a deposit on their first home.
Find out more about this amazing price and enter the competition here.
They were prevented from falling back to their long-term relationship with wages during the financial crisis slump by ultra-low interest rates.
I think house prices are now being maintained at unaffordable levels by a concoction of artificially low mortgage rates thanks to Bank of England and government support and, in the case of Help to Buy, effectively free money.
I also think the measures being taken now to prop up the property market are misguided. And I doubt that the rises we are seeing from a point where houses are already overpriced is going to do the economy any good in the long run and we'll probably see property values rise for some years before coming a cropper again.
But none of that convinces me that over the long-term buying your own home with an affordable repayment mortgage remains anything but a wise move. And to do that, you have to buy a house.
So what can you do when weighing up a home purchase?
You could wait and hope prices fall - or your wages rise substantially.
This involves an element of putting your life on hold, however, and it's still a gamble, as property prices have a tendency to manage to keep on rising even when common sense says they shouldn't.
Alternatively, you could think long-term and try to build in the best margin of safety possible.
That means not panic buying, ditching any thoughts of 'I must buy now or I'll miss the boat', and asking yourself some serious questions about how things stack up.
To that end here are the six things I think are essential to ask yourself.
1. Can I afford my mortgage with interest rates 3% higher?
2. Could I live here for at least five years?
3. Can I add some value?
4. If I let it would the rent more than cover the mortgage?
5. What's the price per square metre and how does that measure up against other properties I'm looking at?
6. Can I negotiate a lower price?
Essentially, they boil down to a series of measures of how much your home would be at risk if your personal finances, employment or the property market took a turn for the worst.
They combine some tests on ability to pay, the home's suitability, value and potential and also the emergency exit strategy of checking it would rent for more than the mortgage.
Hopefully, most of the safety measures will never need to be called upon and the added value bits will pay off.
How would you stress-test a home purchase?
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text:
NIIT National Industry Targeted Aptitude Test
Over the decade, NITAT is an established brand targeted at College Students and Graduates across all academic streams, and close to a million students have benefited from this.
The National IT Aptitude Test (NITAT) has been designed to help individuals who are about to join the workforce to check their Aptitude for new age careers in I.T., Banking , Analytics, Digital Marketing, e-Commerce , Retail and BPM . For the industry, it provides a stream of people who become aware of their capabilities and therefore encourages them to join the industry that best suits their Aptitude.
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For More details visit :www.niit.com/NITAT2015


text:
Bionicle Heroes (GameCube)
Published by Developed by Released Also For |
ESRB Rating Genre Perspective Theme Misc |
Critic Score
100 point score based on reviews from various critics. 4.5
User Score
5 point score based on user ratings. |
User Reviews
There are no reviews for this game.
Our Users Say
Category | Description | User Score |
Acting | The quality of the actors' performances in the game (including voice acting). | 4.5 |
AI | How smart (or dumb) you perceive the game's artificial intelligence to be | 4.5 |
Gameplay | How well the game mechanics work (player controls, game action, interface, etc.) | 4.5 |
Graphics | The quality of the art, or the quality/speed of the drawing routines | 4.5 |
Personal Slant | How much you personally like the game, regardless of other attributes | 4.5 |
Sound / Music | The quality of the sound effects and/or music composition | 4.5 |
Story / Presentation | The main creative ideas in the game and how well they're executed | 4.5 |
Overall User Score (2 votes) | 4.5 |
Critic Reviews
MobyRanks are listed below. You can read here for more information about MobyRank.56
Cheat Code Central
Bionicle is definitely a family-oriented game. There is no blood and not even a robot gets killed. This game is definitely aimed at the young and inexperienced player. Serious gamers need not apply.
Bionicle Heroes is without a doubt aimed at kids and is as E10+ as a shooter can get, but even a child will quickly grow bored by the lack of challenge, innovation, and story development in this game. The blatant similarities to the Lego Star Wars series are also troubling because they often leave you with the feeling that you're playing a retextured version of those games. Although the game does have bursts of inspiration in the charm of the cutscenes, these are spread entirely too thin and don't come close to carrying the package or making up for the disappointing gameplay. Those who enjoy the Bionicle storyline will most likely have an easier time looking past this game's flaws, but if you're looking for a fun and engaging game, definitely pass on this one.
This is not a good game, there are to many disappointing features to warrant purchase and I swear I got a headache at one point from the loud graphics (I had to turn down the brightness on my TV). You may have a big-time Bionicle fan in your house, but you won't be doing them any favors by getting them this title.
Ce n'est pas parce que Bionicle Heroes reprend les mêmes ingrédients que le surprenant LEGO Star Wars II : La Trilogie Originale que le jeu nous plaira à coup sûr. Traveller's Tales s'est planté en optant pour une caméra à l'épaule foireuse, une visée automatique brouillonne, des phases de jeu répétitives et une facilité omniprésente. Bref, Bionicle Heroes aura bien du mal à trouver sa place au pied du sapin à Noël.
Game Freaks 365
Overall, I can't say Bionicle Heroes is the biggest disappointment of the year because I was not expecting much. I think it's safe to say that the game proved to be more disastrous than even I anticipated. It may be mildly entertaining for children, only due to its simplicity, but this is a far cry from the types of games that kids played when I was younger. The challenges of Super Mario Bros. and Sonic the Hedgehog have been replaced with what amounts to automatic success for today's generation of gamers. It's a pity that the developers put such little faith into their customer's abilities, as this game only enables a generation to grow incompetent.


text:
Screenshot Details
The most recent screenshot for this website was taken on Aug 14th, 2013.
We check this website for updates periodically. When we checked 241 days ago, we did not see any significant changes.
We have 22 historic screenshots of Frag-Mutti.de dating back to Jul 7th, 2006.
Our earliest WHOIS history record for the Frag-Mutti.de domain was first recorded in the year 2006.
The registrant on the WHOIS record for this domain is associated with about 59,440 domains.
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adidas Springblade Available Now on miadidas
The bold gets bolder now that the adidas Springblade is available on miadidas. Fans of the slash soled sneakers can customize their own colorway and...More
2The bold gets bolder now that the adidas Springblade is available on miadidas. Fans of the slash soled sneakers can customize their own colorway and...More
Ian Stonebrook 2The adidas Springblade's revolutionary blades go blue on the newest SKU. One third of the recently released "Heather" collection, the adidas Springblade "Heather" Black/Metallic Silver-Solar...More
Jordan Howenstine 0Since its debut last summer, adidas' revolutionary bladed runner has been rolled out in mostly loud colorways. From vivid reds and blues to volt, the...More
Jordan Howenstine 0If the design and appearance of the adidas Springblade wasn't attention grabbing enough already, now the innovative runner is releasing in a bright and loud...More
Jordan Howenstine 2If you've been waiting on a new hue, the adidas Springblade is now available in blue. The revolutionary runner from the Three Stripes was first...More
Jordan Howenstine 0It's been almost two months since the adidas Springblade first hit shelves, and it is fair to say that the innovative design has proven successful....More
Jordan Howenstine 4The adidas Springblade presented intriguing technology and caused a stir when it was released in August, due both to its visual appearance and performance capabilities....More
Jordan Howenstine 2The daring design of the adidas Springblade has everyone interested. While the revolutionary blades have heads turning, many are wondering how they are to run...More
Ian Stonebrook 1Made in Austin


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Philippines Hotel Bookings | Genuine ED Medication | Puerto Galera Hotel Bookings | Subic Bay Hotel Bookings | Viagra Cialis Kamagra | Cheap Airline Tickets | Live LBFM Chat
Toggle shoutbox Philippines Addicts Shoutbox
Dia Filipina Girls That Give Their Occupation As Freelancer
This ad is here because Agoda has changed their policies on hotel bookings regarding affiliates. Agoda is now completely deleting the 365 day cookie along with removing any subsequent
bookings brought though your bookings. Philippines Addicts used to get credit for this along with anybody booking through Agoda 365 days after they initially clicked on our link.
This has
obviously resulted in us losing a substantial amount of money on a monthly basis used to pay our ever increasing server fees (We have two dedicated servers) along with numerous
online marketing campaigns allowing this site to grow by an average of 30 verified people per day. With this said, we ask you click on any of these Agoda links when making a booking
through Agoda so we can get credit for your booking. It costs you nothing and helps us out a lot.
We appreciate your understanding to this truly unfortunately fact. Please click
on these
Agoda links to help Philippines Addicts get credit where it is due, thanks! Book your Philippines Hotel Here and Now!
Posted 08 January 2013 - 11:00 PM
Freelancer in the Philippines means they Self Employed and take work on Ad Hoc when offered to do something for a client.
Posted 11 January 2013 - 04:47 PM
I have been using DIA for the last month (in prep for my Feb trip) and have not come across any profiles that stated they were hookers. however i have collected cell numbers and FB details for a number that are willing to meet 'for a good time'
Posted 15 January 2013 - 01:48 AM
Not surprised, many bar girls and Freelance sex workers on DIAYou ever notice that a lot of those girls on those sites have pictures from hotel lobbies, hotel pools , inside hotel rooms, on a hotel balcony, or sitting on bed thats probably in a hotel. But I'm sure it's just a coincidence.
Posted 24 January 2013 - 12:15 AM
oh well hard work but someones got to do it!
how can you tell other than if they have phone number on their profile.I have gathered some good leads but dont know which order to try and meet them,not sure which will actually meet and which will not look like their pictures.
Posted 27 January 2013 - 01:49 PM
Thats very true those numbers do come in handyI agree the big clue is those girls that have their phone number listed.
I have been using DIA for the last month (in prep for my Feb trip) and have not come across any profiles that stated they were hookers. however i have collected cell numbers and FB details for a number that are willing to meet 'for a good time'
Also tagged with one or more of these keywords: dia, freelancer
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Top Definition
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Minha reserva
This unique city is awaiting your discovery and there's no better way to start exploring than from the comfortable room of your San Francisco hotel. Market Street and the Civic Center are all right around the corner from your San Francisco SOMA hotel. If you're in town for business at the Moscone Center, a conference or convention, you're in the center of the city here. AT&T® Park, Alcatraz, Fisherman's Wharf, Pier 39, Castro District, Golden Gate Bridge, Coit Tower, Lombard Street, the cable cars and Union Square all cause a big conundrum. Where do guests of this San Francisco hotel start? Maybe checking out a San Francisco Giants game is in order. There are several theater and concert venues nearby, so why not check out a show? There are also major shopping areas and restaurants within distance that demand a little attention. Proximity to local attractions and transportation is just one reason why guests keep returning to BEST WESTERN PLUS Americania. The Moscone Center, where notable organizations including Apple and Oracle® host their annual conventions, draws a big crowd. Of course, the 49ers also bring in guests from around the country to nearby Candlestick Park. Checking out a show at SHN® Orpheum Theatre or SHN® Curran Theatre is a real treat. Special events like the 34th America's Cup World Series, and Louis Vuitton Cup have helped define the city. Many guests of this San Francisco hotel are in town for Bay to Breakers, SF Pride Parade, Folsom Street Fair®, or the Chinese New Year Parade. The Marina, Pacific Heights, Mission Bay, and the Financial District also keep guests coming. Business travelers are delighted with our comfortable guest rooms and complimentary high-speed Internet. This San Francisco hotel regularly hosts employees of the San Francisco Federal Building, U.S. Court of Appeals, Twitter®, Sega®, CNET Networks, Dropbox®, BitTorrent®, Yelp, Zynga®, Dolby®, Pintrest and Advent Software. Why not also check out the sights like The Embarcadero or Golden Gate Park at the end of the work day? Book your room at BEST WESTERN PLUS Americania today!
0.02 mile(s) from Federal Building
0.02 mile(s) from 9th Circuit Court
0.30 mile(s) from Civic Center, City Hall, Asian Art Museum, Public Library, Symphony Hall, San Francisco Ballet, War Memorial Auditorium
0.60 mile(s) from Moscone Convention Center, Convention Center and adjacent Moscone West Facility
0.60 mile(s) from Museum of Modern Art, located Near Moscone Center and Yerba Buena Gardens
0.60 mile(s) from Yerba Buena Gardens, park, gardens, shops and museums located near Moscone Center and Museum of Modern Art
0.60 mile(s) from Metreon, movies, shops, restaurants, bars
0.60 mile(s) from Union Square Shopping, independent stores and restaurants as well as Westfield Shopping Center with Nordstrom and Bloomingdales
0.80 mile(s) from Chinatown, shopping, restaurants, historical value
1.00 mile(s) from Bay Bridge, bridge to Oakland/East Bay
1.20 mile(s) from AT&T Park, home of the San Francisco Giants
2.00 mile(s) from Fishermans Wharf, shopping, restuarants, aquarium, historical value
3.00 mile(s) from Japanese Tea Garden, located in Golden Gate Park near the DeYoung Museum
3.00 mile(s) from Golden Gate Park, city's main park with biking, bison, lake, relaxing
4.00 mile(s) from Golden Gate Bridge, bridge to Sausilito/Marin
0.03 mile(s) from Westfield SF Centre, Bloomingdale's, Nordstrom and many other stores at the Westfield SF Centre
0.06 mile(s) from Union Square, Macy's, Neiman Marcus and other world class shopping
1.00 mile(s) from University of California San Francisco, Mission Bay Campus and BioTech Park
10.00 mile(s) from University of California, located in Berkeley, across the Golden Gate Bridge
Choose from a wide selection of restaurants near this San Francisco, California hotel.
Custom Burger & Lounge, on premises, cafe, American, open for: breakfast, lunch, dinner, 7:00 a.m. to 10:00 p.m., all week.
Across the street from Good Pizza, cafe, American, open for: breakfast, lunch, dinner,
121 7th Street
San Francisco, United States


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Bandung's Historical Buildings
| City Square / Alun-alun | Bank Indonesia Building | Gedung Sate |
| Hagel Eens (Au Bon Marche) | Hotel Savoy Homann | Institut Teknologi Bandung |
| Grand Mosque / Mesjid Raya | Banceuy Prison |
Bandung has plenty of historical buildings. Some are well maintained and interesting to visit (or even a must!), while others are left abandoned. A few of the popular Bandung's historical buildings are listed below. Happy reading!
City Square / Alun-alun
Alun-alun Bandung or Bandung's City Square used to be a main place for people to gather and do outdoor activities. Today it seems to have lost its attractiveness since there are many new places for locals to hang out. Though people still come to Alun-alun, usually their main intention is to pray at the Grand Mosque, which is built right on it.
Click the picture above for bigger photo Bandung's of City Square (Alun-alun).
There is a Tourism Information Center (they said it's a temporary site) which is located in the northern side of the mosque, accessible via Jalan Asia Afrika. This office was established by the Bandung Tourism Department in 1973 and provides tourism information within Bandung and the routes, such as factory outlets, eco and historical tourisms, and many more. You might need to ask around to find this small office. Its operating hours: 9am-5pm on weekdays and 9am-2pm on weekend, closed on public holidays. Telephone: +62 22 4206644 (English and Dutch speaking).
the picture above for bigger photos of the Tourism Information Centre.
Location | : | Jl. Asia Afrika, the same location as Grand Mosque |
Admission charges | : | free |
Facilities | : | toilets, basement parking area |
Nearby facilities | : | ATM, hotels, pharmacies, money changers, post office |
Nearby attractions | : | Museum Asia-Africa, shopping center |
How to get there | : | by angkot: from Jalan Merdeka, take the "Dago-Kalapa" line and stop at Kalapa station (fare: Rp. 3,000 in 20 minutes). From there, take an angkot that goes to the it square (either "Kalapa-Sukajadi" or "Kalapa-Buah Batu"). It costs: Rp. 1,500 and takes only 5 minutes |
Remarks | : | beware of pickpockets around Alun-alun. For more information about Tourism Information Center, you might call Mr. Ajid (the official staff of the local tourism aegy) on the phone number above. |
Bank Indonesia Building
This building used to be named "De Javasche Bank Bandung" during the Dutch colony era. It was built in 1909 facing Kerklaan, which is now known as Jalan Perintis Kemerdekaan. Then Edward Cuypers, a Dutch architect, rebuilt it into a bigger building and finished it in 1918. After gaining its independence in 1945, the Indonesian government took over the building and made it became the building of Bank Indonesia in 1953.
Click the picture above for more and bigger photos of Bank Indonesia Building.
Location | : | Jl. Merdeka and Jl. Wastukencana Junction - click here for location map |
Nearby attractions | : | old buildings, such as church of Bethel and Cathedral, City Hall, and other historical buildings along Jalan Braga |
Nearby facilities | : | ATM/banks, church, shops, hotels, a shopping mall |
How to get there | : | by angkot: from Jalan Merdeka, take the one that goes to Ledeng terminal. After about 3 minutes journey, you can stop right in front of Gedung Bank Indonesia. Other option is by angkot that goes to St. Hall (from Dago terminal), which also stops near Gedung Bank Indonesia. |
Gedung Sate
The building was built in 1920 by a Dutch architect, J.Gerber. It is named Gedung Sate or "Satay building" because of the shape of its main roof and was designed based on a combination of Italian and local architectures, Islamic and Hindu. The "satay roof" consists of 6 "satay-look alike pieces", to symbolize the 6 million Dutch Gulden spent to build Gedung Sate. The Dutch East Indies government used this building as its main office and today Gedung Sate is the office of the West Java Governor.
This building was purposely built facing Gunung (mount) Tangkuban Perahu. Later, the government of Indonesia constructed Monument Perjuangan Rakyat Jawa Barat in front of Gedung Sate. If we draw a line from Gunung Tangkuban Perahu to Monument Perjuangan Rakyat Jawa Barat and to Gedung Sate, it will be a straight line. These three sites are all located on the same latitude.
Click the picture above for bigger photo of Gedung Sate.
Location | : | Jl. Diponegoro No. 22 - click here for location map | Admission charges | : | free | Facilities | : | toilets | Nearby facilities | : | mini markets, pharmacy, ATMs, hotel | Nearby attractions | : | Museum Geologi, Museum Pos Indonesia | How to get there | : | On foot from Jalan Ir. H. Juanda (Dago - just to the north of Dago Plaza) then onto Jalan Diponegoro approx. 900 meters, from the junction of Jalan R.E.Martadinata (Riau) and Jalan Banda approximately 1 kilometer [0.62 miles], from Jalan Merdeka (Bandung Indah Plaza) approximately 1.7 kilometers [1.06 miles]
By angkot: from the intersection of Jl. Ir. H. Juanda and Jl. Sulanjana, take an angkot that goes to Cicaheum and get off right in front of Gedung Sate (fare: Rp. 2,000, only about 5 minutes away). Please refer to the location map too |
Remarks | : | Gedung Sate is open for pulic visitors only on weekends and public holidays, and a prior arrangement with Mr. Wawan (internal security staff of Gedung Sate) is required. Every Sunday, there is a Sunday market in front of Gedung Sate (at the Gasibu area). This attracts many local residents and causes traffic jam around the area too. |
Hagel Eens (Au Bon Marche)
In 1930, this building used to be one of the most famous trendy fashion shops. The shop used to sell expensive and elegant fashion products from France, owned by Au Bon Marche. Before that, this building was owned by Hagel Eens family, who also sold fashion products but later decided to sell their shop to Au Bon Marche. Au Bon Marche run the shop without erasing Hagel Eens family name, which still can be seen on the wall of today's building. Currently the building is currently under a major renovation.
Click the picture above for bigger photo of Hagel Eens (Au Bon Marche).
Location | : | Jl. Braga |
Nearby facilities | : | pharmacies, money changer, hotels, ATMs, Tourist Information Center, restaurants, souvenir shops |
Nearby attractions | : | local art shops, paintings, Grand Mosque / Mesjid Raya, Museum Asia Africa, Alun-Alun, Braga City Walk |
How to get there | : | walk about 300 meters (328 yards) from the Alun-alun towards the north along Jalan Braga |
Remarks | : | today this building becomes a cafe. |
Hotel Savoy Homann
This hotel was initially named Hotel Homann. It was designed by a Dutch architect named Albert F. Aalbers and was owned by the Homann family in the late 1800s. Homann was a German immigrant who arrived in the 1870's. When the new building was completed it was then renamed Hotel Savoy in 1939.
Click the picture aove for more and bigger photos of the Hotel Savoy Homann.
It has subsequently been recognized as one of the city's landmark buildings for its tropical art deco architecture.
Hotel Homann or Savoy or Savoy Homann has had famous people on its guest list, such as the King and Queen of Siam (Thailand) in the late 1890's, Charlie Chaplin and Mary Pickford who stayed in 1927, Indian Prime Minister PJ Nehru, Egyptian President Gamal Abdul Naseer in 1955, The Duchess of Westminster, and so on. Charlie Chaplin even visited more than once.
Location | : | Jl. Asia Afrika No. 112 (a few hundred meters east of Mesjid Raya) |
Telephone | : | +62 22 4232244 |
Fax | : | +62 22 4236187 |
: | firstname.lastname@example.com, firstname.lastname@example.com | |
Website | : | www.savoyhomann-hotel.com |
Facilities | : | Wi-Fi, restaurant, bar & lounge, ballroom, meeting room, wellness & spa, swimming pool, karaoke room, business center, drugstore, games room, pick up resvice from/to airport or train station are available |
Nearby facilities | : | ATMs/banks, Bandung main train station, churches, book shop, hospital/clinic/chemist, mosques, postal services, supermarkets |
Nearby attractions | : | Jl. Braga, City Hall, Museum Konperensi Asia Afrika |
How to get there | : | from Dago Plaza, take angkot that goes to Kelapa Terminal and get off at intersection of Jl. Asia Afrika (fare: Rp. 2,500 for a 15-minute of journey) and walk for approximately 0.08 miles to the hotel |
Remarks | : | this hotel is one of the city's landmark buildings and has 185 rooms. Published room rates start from Rp. 1,200,000. |
Institut Teknologi Bandung
De Techniche Hoogeschool te Bandung was built in 1920 by the Dutch and renamed Institut Teknologi Bandung (ITB) in 1959. Although the university has grown in size, faculties and numbers of students, the original buildings are well maintained.
Institut Teknologi Bandung has a unique roof style which was designed by architect Henri Maclaine-Pont, based on a West Sumatera traditional house, which still can be seen today on its east and west halls.
Click the picture above for more and bigger photo of Institut Teknologi Bandung.
ITB has a view interesting remarks, such as the first president of Indonesia, Soekarno, graduated from this university in the late 1920's. Asiaweek magazine in 2000 ranked ITB ahead of Australian technology universities – such as the Queensland University of Technology, University of Technology Sydney, and so on, and it is now ranked in the 21st position, down from 15th in 1999. In 2004 ITB reportedly had more than 14,000 students.
Location | : | Jl. Ganesha No. 10 |
Telephone | : | +62 22 2500935 |
Website | : | www.itb.ac.id |
Nearby attractions | : | Bandung's Taman Sari Zoo and Zoo Museum |
How to get there | : | By angkot: if you take the "Dago" direction, stop in front of Borromeus hospital near Jalan Ganesha and continue on foot about 600 meters (656 yards), if you take "Sadangserang-Caringin" or "Cicaheum-Ledeng", you can stop right in front of Bandung zoo and ITB is across the road
By taxi: from Jalan Merdeka it costs about Rp. 20,000, from Alun-alun (Jalan Asia Afrika) about Rp. 35,000 |
Remarks | : | tourists are not allowed to enter the university unless prior permission is obtained, however the buildings are visible from the roads. |
Grand Mosque / Mesjid Raya
This recently renovated building (finished in 2004) is the main mosque in Bandung. Bandung's Grand Mosque has two 81-meter-tall [265 feet] minarets. The initial idea was to build two 99-meter [324 feet] minarets because of a religious belief, but since there is an airport nearby, it was not possible to get permission from the local authority (because of an aviation safety reason).
Click the picture above for more and bigger photos of the Bandung's Grand Mosque.
The original mosque itself was built in 1811, made of wood and bamboo, but in 1930 a new mosque was redesigned by Henri Maclaine-Port. The Grand Mosque can accommodate a maximum of 14,000 worshippers. After renovation, date-palm trees were planted around the mosque garden and a Turkish-Mediterranean style fountain was also built in front of the mosque.
You can climb up one of the minarets with a small admission charge of Rp. 2,000. Its operating hours: from 9am to 5 pm. A staff will escort you to the top, which is reach by taking a lift.
Location | : | Jl. Asia Afrika (right on Alun-alun) |
Nearby facilities | : | ATMs, money changer, internet café |
Nearby attractions | : | Museum Asia-Africa, Savoy Homann Hotel, Jalan Braga, "Pikiran Rakyat "Newspaper Building (art-deco building), Monument KM BDG 0+00, Jl. Asia Afrika |
How to get there | : | Within walking distance from Jalan Asia Afrika and Jalan Braga; from Jalan Merdeka (Bandung Indah Plaza, Hyatt hotel, Santika hotel, etc) on foot (approx. 1.2 kilometers or 0.75 miles walk)
By angkot: take angkot that goes to "Kalapa" from "Dago" direction (fare: Rp. 2,500) |
Remarks | : | should you want to see inside the mosque, you will need to dress appropriately, no food or drink is allowed inside and do not walk in front of Muslims who are praying. Shoes must be off before going inside the mosque and you can keep them in the lockers provided. Beware of pickpockets around Alun-alun/Mesjid Raya. |
Banceuy Prison
In 1871, the government of the Dutch East Indies (now Indonesia) built Banceuy Prison to detain political prisoners and criminal. One of the prisoners was the first president of Indonesia, Ir. Soekarno, who spent over 8 months in Banceuy Prison. He was arrested on 29 December 1929 and thrown into a cell located in Block F No. 5. Mostly a cell measured only about 2.5 meters x 1.5 meters with a folding cot and non-permanent toilets inside. During his detention there, he prepared an advocacy "Indonesia Menggugat", which then was read at Landraad Building (now called Indonesia Menggugat Building) on 30 August 1930.
Click the picture above for more and bigger photos of the Banceuy Prison.
In 1983, Banceuy Prison was moved to Jl. Soekarno-Hatta, Bandung. The old "Soekarno prison" building was later being demolished to build a new shopping complex, named "Banceuy Permai", leaving only Soekarno's cell and a tower guard. Unfortunately, this "prison" is currently not in its best condition and since it's covered by a fence, you only can see the prison from far away.
Location | : | Jl. Belakang Factory (to the north of Grand Mosque) |
Nearby facilities | : | ATMs, pharmacy, money changer, hotels |
Nearby attractions | : | City Square, Grand Mosque, Jl. Asia Afrika, Museum Asia-Africa, Monument KM BDG 0+00 |
How to get there | : | Tourist Information Center (located near Grand Mosque), walk approx.0.14 miles or 250 meters to Banceuy Prison. |
...index :Previous page | Next page: Other Interesting Places... |
Your Comments: 1 comment |
Name: |
Dutchman | 20 Oct 2014 | |
Comment: |
Re: Hagel Eens (Au Bon Marché), Jl. Braga, Bandung The name should be: Hagelsteens. This fashion house on Braga Road no. 3 was initially owned by August Hagelsteens, a fashion retailer born in Antwerp, Belgium, who died in Bandung on 22 November 1922 at the age of 52 after a serious illness. The fashion house Au Bon Marché on Braga Road 3 to 9 was owned by the Makkinga family. The last owner, A. Makkinga, died in Amsterdam, The Netherlands, on 28 May 1955 at the age of 72. He lived in Bandung from 1911 until 1952. The Hagelsteens and Makkinga families of Braga Road were very popular in Bandung and made significant contributions to the development of the city. See also: http://kranten.delpher.nl http://kranten.delpher.nl See also: |


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Media reports recently indicated that Copa Airlines is planning to aim for a slightly more modest ASK growth of 14 percent in 2013. The reports also stated that Panama's flagship carrier Copa Airlines will be focusing more on adding capacity to its existing markets this year. Copa Airlines has only added Boston as its new destination so far this year. This only destination to be added on the airline's network was introduced on July 11, 2013. The airline has since been operating one daily flight to Boston. Aimed at including more destinations on its network, Copa Airlines has announced that it will be launching flights to Tampa starting from December 16, 2013. The airline will be operating four flights every week on this route. With Boston and Tampa on its flight network now this year, Copa Airlines will be offering services to 66 destinations. The airline will be serving 63 destinations from the Panamanian capital Panama City, and nine destinations in the United States. Copa Airlines said that its ASKs increased 19 percent y-o-y in H1 2013. The airline further said that its RPKs were up 20 percent during the same period this year, while its load factor also improved by 0.7 point to 76 percent. According to the airline, the H1 results are a strong indication of the buoyant demand in the intra-Latin American market. Copa Airlines succeeded in its capacity expansion this year with the help of seven newly purchased Boeing 737-800 aircrafts. In 2013, the airline has so far 83 aircrafts in its fleet and plans to increase it to 90 aircrafts by the end of this year.


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Fred Emilio Zweig
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Grand Portage National Monument
Wolves Wander From Grand Portage Indian Reservation To Isle Royale National Park And Back
A pair of wolves whose home range is on the Grand Portage Indian Reservation in Minnesota recently used an ice bridge to cross 14 miles over Lake Superior to Isle Royale National Park, where they wandered for five days before returning to the mainland.
Paddling The Border Route In The Boundary Waters
It's a 200-mile paddle along Minnesota's Border Route, from Crane Lake on the eastern side of Voyageurs National Park to Grand Portage National Monument on Lake Superior. There are twists and turns as it follows a series of pristine, remote lakes linked by portages along the Minnesota and Ontario border.
Dispersing Isle Royale National Park Wolf Killed By Pellet Wound
A female wolf that crossed from Isle Royale National Park to Grand Portage National Monument via an ice bridge across Lake Superior was killed by a pellet gun wound, according to veterinarians.
Via A Wayward Wolf, Connections Strengthened Between Isle Royale And Grand Portage
Recently, a wolf left Isle Royale National Park for the mainland via an ice bridge across Lake Superior. It later was found dead in Grand Portage National Monument. What follows is a story about the connections between Isle Royale and Grand Portage.
Grand Portage National Monument Altering Schedule To Cope With Budget Cuts
A slight shift in operational hours at Grand Portage National Monument in Minnesota will occur late this spring due to budget cuts mandated by Congress and the White House.
Interpretive Film At Grand Portage National Monument Includes Version In Ojibwemowin
In what may be a first, an interpretive film created for Grand Portage National Monument in Minnesota is available in Ojibwemowin, the traditional language of the Grand Portage people.
Traveler's Checklist: Grand Portage National Monument
Wilderness exploration, high finance, and a frontier trading post are all part of the story at Grand Portage National Monument (Minnesota), and the park's big annual event is coming up later this week. Here are some tips to make the most of your visit, summer or fall.
Minnesota's Grand Portage National Monument Commemorates the Historic Fur Trade Era
Located on an Indian reservation in northeastern Minnesota, Grand Portage National Monument was established September 15, 1951, to commemorate the historic North American fur trade. A British fur trading company operated a summer headquarters and western supply depot at Grand Portage from 1778 until 1802.
Apostle Islands, Pictured Rocks National Lakeshores Taking Steps to Prevent Spread of Fish Virus
Fishing in waters of Apostle Islands and Pictured Rocks national lakeshores is going to be a bit more difficult this year, as emergency restrictions are being implemented in an effort to prevent the spread of a deadly fish virus.
Parks and Tribe Establish Plan to Fight Fish Disease in Lake Superior
The National Park Service and the Grand Portage Band of the Lake Superior Chippewa have agreed to work together on efforts to protect park and tribal fishery resources in Lake Superior from a deadly fish disease known as viral hemorrhagic septicemia, or VHS.
A New Foundation Arises to Aid Lake Superior Parks
As times grow increasingly tougher, financially, for the National Park Service, individual parks can find a bit of a lifeline in non-profit friends groups. The latest non-profit to surface is the National Parks of Lake Superior Foundation.


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A 4-CD unabridged audiobook of The Lion, the Witch and the Wardrobe, book two in the classic fantasy series, The Chronicles of Narnia. Narrated by renowned actor Michael York, you can also put the CDs in your computer to view a gallery of classic full-color illustrations by the original illustrator of Narnia, Pauline Baynes.
Four adventurous siblings--Peter, Susan, Edmund, and Lucy Pevensie-- step through a wardrobe door and into the land of Narnia, a land frozen in eternal winter and enslaved by the power of the White Witch. But when almost all hope is lost, the return of the Great Lion, Aslan, signals a great change . . . and a great sacrifice.
Open the door and enter a new world with The Lion, the Witch and the Wardrobe, the second book in C. S. Lewis's classic fantasy series, which has captivated readers of all ages for over sixty years. This is a stand-alone novel, but if you want to return to Narnia, read The Horse and His Boy, the third book in The Chronicles of Narnia.


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DisuniformityJason RantanenUniversity of Iowa - College of Law Lee PetherbridgeLoyola Law School Los Angeles January 24, 2014 U Iowa Legal Studies Research Paper No. 13-42 Loyola-LA Legal Studies Paper No. 2013-39 Abstract: The Federal Circuit is a response to a failure in judicial administration that produced a fractured, unworkable patent law; one that Congress concluded ill-served entrepreneurship and innovation. The purpose of the response – vesting exclusive jurisdiction for patent appeals in the Federal Circuit – was to permit that court to develop patent law in the direction of greater clarity and uniformity. Both at the time of the Federal Circuit's creation and more recently, scholars, judges, and practitioners have waged great debates over whether patent law uniformity furthers the ultimate goals of entrepreneurship and innovation. These debates have rested on a largely untested empirical proposition: that the Federal Circuit's patent law jurisprudence embodies a move towards doctrinal uniformity. This paper reports an empirical study that examines patent law uniformity through the measure of open decisional disagreement between Federal Circuit judges. Its central empirical observation is a remarkable increase in decisional disagreement – indicative of a decline in doctrinal uniformity – among Federal Circuit judges over the past several years. The paper raises and discusses several possible explanations for its surprising observations, including, inter alia, the Supreme Court and personnel changes at the Federal Circuit. It also considers what the observations and explanations might contribute to a current debate over the merits of Congress's decision to unify patent jurisdiction in the Federal Circuit. Number of Pages in PDF File: 40 Keywords: patent, empirical, Federal Circuit Date posted: November 11, 2013 ; Last revised: February 4, 2014Suggested CitationContact Information
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