bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Definition of Vaginal Microbiota in East African Commercial Sex Workers
Authors: Schellenberg John J. , Links Matthew G. , Hill Janet E. , Dumonceaux Tim J. , Kimani Joshua , Jaoko Walter , Wachihi Charles , Mungai Jane Njeri , Peters Geoffrey A. , Tyler Shaun , Graham Morag , Severini Alberto , Fowke Keith R. , Ball T. Blake , Plummer Francis A.
Published: April 2011

Resistance to HIV infection in a cohort of commercial sex workers living in Nairobi, Kenya, is linked to mucosal and antiinflammatory factors that may be influenced by the vaginal microbiota. Since bacterial vaginosis (BV), a polymicrobial dysbiosis characterized by low levels of protective
Lactobacillus
organisms, is an established risk factor for HIV infection, we investigated whether vaginal microbiology was associated with HIV-exposed seronegative (HESN) or HIV-seropositive (HIV
+
) status in this cohort. A subset of 44 individuals was selected for deep-sequencing analysis based on the chaperonin 60 (
cpn60
) universal target (UT), including HESN individuals (
n
= 16), other HIV-seronegative controls (HIV-N,
n
= 16), and HIV
+
individuals (
n
= 12). Our findings indicate exceptionally high phylogenetic resolution of the
cpn60
UT using reads as short as 200 bp, with 54 species in 29 genera detected in this group. Contrary to our initial hypothesis, few differences between HESN and HIV-N women were observed. Several HIV
+
women had distinct profiles dominated by
Escherichia coli
. The deep-sequencing phylogenetic profile of the vaginal microbiota corresponds closely to BV
+
and BV
−
diagnoses by microscopy, elucidating BV at the molecular level. A cluster of samples with intermediate abundance of
Lactobacillus
and dominant
Gardnerella
was identified, defining a distinct BV phenotype that may represent a transitional stage between BV
+
and BV
−
. Several alpha- and betaproteobacteria, including the recently described species
Variovorax paradoxus
, were found to correlate positively with increased
Lactobacillus
levels that define the BV
−
(“normal”) phenotype. We conclude that
cpn60
UT is ideally suited to next-generation sequencing technologies for further investigation of microbial community dynamics and mucosal immunity underlying HIV resistance in this cohort.
Novel (2
R
,3
R
)-2,3-Butanediol Dehydrogenase from Potential Industrial Strain Paenibacillus polymyxa ATCC 12321
Authors: Yu Bo , Sun Jibin , Bommareddy Rajesh Reddy , Song Lifu , Zeng An-Ping
Published: April 2011

A (2
R
,3
R
)-2,3-butanediol dehydrogenase (BDH99::67) from
Paenibacillus polymyxa
ATCC 12321 was functionally characterized. The genetic characteristics of BDH99::67 are completely different from those of
meso
- and (2
S
,3
S
)-2,3-butanediol dehydrogenases. The results showed that BDH99::67 belongs to the medium-chain dehydrogenase/reductase superfamily and not to the short-chain dehydrogenase/reductase superfamily, to which
meso
- and (2
S
,3
S
)-2,3-butanediol dehydrogenases belong.
Comparison of Two Generations of the Panbio Dengue NS1 Capture Enzyme-Linked Immunosorbent Assay
Authors: Lima Monique da Rocha Queiroz , Nogueira Rita Maria Ribeiro , Bispo de Filippis Ana Maria , dos Santos Flavia Barreto
Published: April 2011

We compared two generations of Panbio (Brisbane, Australia) commercial kits for NS1 antigen capture for early diagnosis of dengue: the first-generation pan-E Dengue Early ELISA and the second-generation Dengue Early ELISA. The test improvement resulted in a highly sensitive and specific test suitable for use as a first-line test in the field.
Paxillin and Phospholipase D Interact To Regulate Actin-Based Processes in Dictyostelium discoideum
Authors: Pribic Jelena , Garcia Rebecca , Kong May , Brazill Derrick
Published: April 2011

The actin cytoskeleton forms a membrane-associated network whose proper regulation is essential for numerous processes, including cell differentiation, proliferation, adhesion, chemotaxis, endocytosis, exocytosis, and multicellular development. In this report, we show that in
Dictyostelium discoideum
, paxillin (PaxB) and phospholipase D (PldB) colocalize and coimmunoprecipitate, suggesting that they interact physically. Additionally, the phenotypes observed during development, cell sorting, and several actin-required processes, including cyclic AMP (cAMP) chemotaxis, cell-substrate adhesion, actin polymerization, phagocytosis, and exocytosis, reveal a genetic interaction between
paxB
and
pldB
, suggesting a functional interaction between their gene products. Taken together, our data point to PldB being a required binding partner of PaxB during processes involving actin reorganization.
Calcineurin Controls Drug Tolerance, Hyphal Growth, and Virulence in Candida dubliniensis
Authors: Chen Ying-Lien , Brand Alexandra , Morrison Emma L. , Silao Fitz Gerald S. , Bigol Ursela G. , Malbas Fedelino F. , Nett Jeniel E. , Andes David R. , Solis Norma V. , Filler Scott G. , Averette Anna , Heitman Joseph
Published: April 2011

Candida dubliniensis
is an emerging pathogenic yeast species closely related to
Candida albicans
and frequently found colonizing or infecting the oral cavities of HIV/AIDS patients. Drug resistance during
C. dubliniensis
infection is common and constitutes a significant therapeutic challenge. The calcineurin inhibitor FK506 exhibits synergistic fungicidal activity with azoles or echinocandins in the fungal pathogens
C. albicans
,
Cryptococcus neoformans
, and
Aspergillus fumigatus
. In this study, we show that calcineurin is required for cell wall integrity and wild-type tolerance of
C. dubliniensis
to azoles and echinocandins; hence, these drugs are candidates for combination therapy with calcineurin inhibitors. In contrast to
C. albicans
, in which the roles of calcineurin and Crz1 in hyphal growth are unclear, here we show that calcineurin and Crz1 play a clearly demonstrable role in hyphal growth in response to nutrient limitation in
C. dubliniensis
. We further demonstrate that thigmotropism is controlled by Crz1, but not calcineurin, in
C. dubliniensis
. Similar to
C. albicans
,
C. dubliniensis
calcineurin enhances survival in serum.
C. dubliniensis
calcineurin and
crz1/crz1
mutants exhibit attenuated virulence in a murine systemic infection model, likely attributable to defects in cell wall integrity, hyphal growth, and serum survival. Furthermore, we show that
C. dubliniensis
calcineurin mutants are unable to establish murine ocular infection or form biofilms in a rat denture model. That calcineurin is required for drug tolerance and virulence makes fungus-specific calcineurin inhibitors attractive candidates for combination therapy with azoles or echinocandins against emerging
C. dubliniensis
infections.
Differential Chitinase Activity and Production within Francisella Species, Subspecies, and Subpopulations
Authors: Chandler Jeffrey C. , Molins Claudia R. , Petersen Jeannine M. , Belisle John T.
Published: April 2011

Genotyping of
Francisella tularensis
(A1a, A1b, A2, and type B) and
Francisella novicida
has identified multiple differences between species and among
F. tularensis
subspecies and subpopulations. Variations in virulence, geographic distribution, and ecology are also known to exist among this group of bacteria, despite the >95% nucleotide identity in their genomes. This study expands the description of phenotypic differences by evaluating the ability of
F. tularensis
and
F. novicida
to degrade chitin analogs and produce active chitinases. Endochitinase activities were observed to vary among
F. tularensis
and
F. novicida
strains. The activity observed for
F. tularensis
strains was predominantly associated with whole-cell lysates, while the chitinase activity of
F. novicida
localized to the culture supernatant. In addition, the overall level of chitinase activity differed among the subpopulations of
F. tularensis
and between the species. Bioinformatic analyses identified two new putative chitinase genes (
chiC
and
chiD
), as well as the previously described
chiA
and
chiB
. However, the presence of these four open reading frames as intact genes or pseudogenes was found to differ between
Francisella
species and
F. tularensis
subspecies and subpopulations. Recombinant production of the putative chitinases and enzymatic evaluations revealed ChiA, ChiB, ChiC, and ChiD possessed dissimilar chitinase activities. These biochemical studies coupled with bioinformatic analyses and the evaluation of
chiA
and
chiC
knockouts in
F. tularensis
A1 and A2 strains, respectively, provided a molecular basis to explain the differential chitinase activities observed among the species and subpopulations of
Francisella
.
PtrA Is a Periplasmic Protein Involved in Cu Tolerance in Pseudomonas aeruginosa
Authors: Elsen Sylvie , Ragno Michel , Attree Ina
Published: April 2011

In this work, we demonstrate that PtrA (U. H. Ha et al., Mol. Microbiol. 54:307–320, 2004) is a periplasmic protein, specifically synthesized in the presence of copper, that is involved in the tolerance of
Pseudomonas aeruginosa
to copper. Our biochemical and genetic analyses challenge its role in transcriptional inhibition of the type III secretion system.
Complete Genome Sequence and Immunoproteomic Analyses of the Bacterial Fish Pathogen Streptococcus parauberis
Authors: Nho Seong Won , Hikima Jun-ichi , Cha In Seok , Park Seong Bin , Jang Ho Bin , del Castillo Carmelo S. , Kondo Hidehiro , Hirono Ikuo , Aoki Takashi , Jung Tae Sung
Published: April 2011

Although
Streptococcus parauberis
is known as a bacterial pathogen associated with bovine udder mastitis, it has recently become one of the major causative agents of olive flounder (
Paralichthys olivaceus
) streptococcosis in northeast Asia, causing massive mortality resulting in severe economic losses.
S. parauberis
contains two serotypes, and it is likely that capsular polysaccharide antigens serve to differentiate the serotypes. In the present study, the complete genome sequence of
S. parauberis
(serotype I) was determined using the GS-FLX system to investigate its phylogeny, virulence factors, and antigenic proteins.
S. parauberis
possesses a single chromosome of 2,143,887 bp containing 1,868 predicted coding sequences (CDSs), with an average GC content of 35.6%. Whole-genome dot plot analysis and phylogenetic analysis of a 60-kDa chaperonin-encoding gene and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-encoding gene showed that the strain was evolutionarily closely related to
Streptococcus uberis. S. parauberis
antigenic proteins were analyzed using an immunoproteomic technique. Twenty-one antigenic protein spots were identified in
S. parauberis
, by reaction with an antiserum obtained from
S. parauberis
-challenged olive flounder. This work provides the foundation needed to understand more clearly the relationship between pathogen and host and develops new approaches toward prophylactic and therapeutic strategies to deal with streptococcosis in fish. The work also provides a better understanding of the physiology and evolution of a significant representative of the
Streptococcaceae
.
Characterization of CrgA, a New Partner of the Mycobacterium tuberculosis Peptidoglycan Polymerization Complexes
Authors: Plocinski P. , Ziolkiewicz M. , Kiran M. , Vadrevu S. I. , Nguyen H. B. , Hugonnet J. , Veckerle C. , Arthur M. , Dziadek J. , Cross T. A. , Madiraju M. , Rajagopalan M.
Published: April 2011

The role(s) in cell division of the
Mycobacterium tuberculosis
Rv0011c gene product, a homolog of the
Streptomyces
CrgA protein that is responsible for coordinating growth and cytokinesis in sporogenic aerial hyphae, is largely unknown. We show that an enhanced cyan fluorescent protein-
M. tuberculosis
CrgA (ECFP-CrgA
MT
) fusion protein is localized to the cell membrane, midcell, and cell pole regions in
Mycobacterium smegmatis
. Furthermore, the ECFP-CrgA
MT
fusion protein colocalized with FtsZ-enhanced yellow fluorescent protein (EYFP) in
M. smegmatis
. Bacterial two-hybrid assays indicated strong interactions of
M. tuberculosis
CrgA with FtsZ, FtsQ, and the class B penicillin-binding proteins, FtsI (PBPB) and PBPA. The midcell localization of CrgA
MT
was severely compromised under conditions of FtsZ depletion, which indicated that CrgA localizes to the midcell region after assembly of the FtsZ ring.
M. tuberculosis
cells with reduced CrgA levels were elongated and grew more slowly than wild-type cells, which indicated defects in cell division, whereas CrgA overproduction did not show growth defects. A
M. smegmatis
Δ
crgA
strain exhibited a bulged cell morphology, elongated cells with a chain-like phenotype, cells with polar bulbous structures, and a modest growth defect. FtsZ and FtsI levels were not affected in cells producing altered levels of CrgA. Septal and membrane localization of GFP-FtsI was enhanced by CrgA overproduction and was diminished in a Δ
crgA
strain, which indicates that one role of CrgA is to promote and/or stabilize FtsI localization. Overall, these data indicate that CrgA is a novel member of the cell division complex in mycobacteria and possibly facilitates septum formation.
Roles of Cysteine Proteases Cwp84 and Cwp13 in Biogenesis of the Cell Wall of Clostridium difficile
Authors: de la Riva Lucía , Willing Stephanie E. , Tate Edward W. , Fairweather Neil F.
Published: April 2011

Clostridium difficile
expresses a number of cell wall proteins, including the abundant high-molecular-weight and low-molecular-weight S-layer proteins (SLPs). These proteins are generated by posttranslational cleavage of the precursor SlpA by the cysteine protease Cwp84. We compared the phenotypes of
C. difficile
strains containing insertional mutations in either
cwp84
or its paralog
cwp13
and complemented with plasmids expressing wild-type or mutant forms of their genes. We show that the presence of uncleaved SlpA in the cell wall of the
cwp84
mutant results in aberrant retention of other cell wall proteins at the cell surface, as demonstrated by secretion of the proteins Cwp66 and Cwp2 into the growth medium. These phenotypes are restored by complementation with a plasmid expressing wild-type Cwp84 enzyme but not with one encoding a Cys116Ala substitution in the active site. The
cwp13
mutant cleaved the SlpA precursor normally and had a wild-type-like colony phenotype. Both Cwp84 and Cwp13 are produced as proenzymes which are processed by cleavage to produce mature enzymes. In the case of Cwp84, this cleavage does not appear to be autocatalytic, whereas in Cwp13 autocatalysis was demonstrated as a Cys109Ala mutant did not undergo processing. Cwp13 appears to have a role in processing of Cwp84 but is not essential for Cwp84 activity. Cwp13 cleaves SlpA in the HMW SLP domain, which we suggest may reflect a role in cleavage and degradation of misfolded proteins at the cell surface.
The Poor Growth of Rhodospirillum rubrum Mutants Lacking RubisCO Is Due to the Accumulation of Ribulose-1,5-Bisphosphate
Authors: Wang Di , Zhang Yaoping , Pohlmann Edward L. , Li Jilun , Roberts Gary P.
Published: April 2011

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) catalyzes the first step of CO
2
fixation in the Calvin-Benson-Bassham (CBB) cycle. Besides its function in fixing CO
2
to support photoautotrophic growth, the CBB cycle is also important under photoheterotrophic growth conditions in purple nonsulfur photosynthetic bacteria. It has been assumed that the poor photoheterotrophic growth of RubisCO-deficient strains was due to the accumulation of excess intracellular reductant, which implied that the CBB cycle is important for maintaining the redox balance under these conditions. However, we present analyses of
cbbM
mutants in
Rhodospirillum rubrum
that indicate that toxicity is the result of an elevated intracellular pool of ribulose-1,5-bisphosphate (RuBP). There is a redox effect on growth, but it is apparently an indirect effect on the accumulation of RuBP, perhaps by the regulation of the activities of enzymes involved in RuBP regeneration. Our studies also show that the CBB cycle is not essential for
R. rubrum
to grow under photoheterotrophic conditions and that its role in controlling the redox balance needs to be further elucidated. Finally, we also show that CbbR is a positive transcriptional regulator of the
cbb
operon (
cbbEFPT
) in
R. rubrum
, as seen with related organisms, and define the transcriptional organization of the
cbb
genes.
Characteristic Phenotypes Associated with
ptsN
-Null Mutants in Escherichia coli K-12 Are Absent in Strains with Functional
ilvG
Authors: Reaves Marshall Louis , Rabinowitz Joshua D.
Published: April 2011

The phosphotransferase system (PTS), encompassing EI, HPr, and assorted EII proteins, uses phosphoenolpyruvate to import and phosphorylate sugars. A paralog of EIIA of the sugar PTS system known as
ptsN
has been purported to regulate organic nitrogen source utilization in
Escherichia coli
K-12. Its known biochemical function, however, relates to potassium homeostasis. The evidence for regulation of organic nitrogen source utilization by
ptsN
is based primarily on the defective growth of Δ
ptsN
mutants on amino acid nitrogen sources and other nutrient combinations. These observations were made with
E. coli
strains MG1655 and W3110, which carry a nonfunctional version of
ilvG
. There are three isozymes that effectively catalyze the first committed step of branched-chain amino acid biosynthesis, but
ilvG
is unique for doing so effectively across a range of potassium concentrations. Here we show that all of the nutrient utilization phenotypes attributed to
ptsN
are manifested selectively in strains lacking functional
ilvG
. We conclude that the
ptsN
gene product does not regulate organic nitrogen source utilization as previously proposed.
Inference of the Transcriptional Regulatory Network in Staphylococcus aureus by Integration of Experimental and Genomics-Based Evidence
Authors: Ravcheev Dmitry A. , Best Aaron A. , Tintle Nathan , DeJongh Matthew , Osterman Andrei L. , Novichkov Pavel S. , Rodionov Dmitry A.
Published: April 2011

Transcriptional regulatory networks are fine-tuned systems that help microorganisms respond to changes in the environment and cell physiological state. We applied the comparative genomics approach implemented in the RegPredict Web server combined with SEED subsystem analysis and available information on known regulatory interactions for regulatory network reconstruction for the human pathogen
Staphylococcus aureus
and six related species from the family
Staphylococcaceae
. The resulting reference set of 46 transcription factor regulons contains more than 1,900 binding sites and 2,800 target genes involved in the central metabolism of carbohydrates, amino acids, and fatty acids; respiration; the stress response; metal homeostasis; drug and metal resistance; and virulence. The inferred regulatory network in
S. aureus
includes ∼320 regulatory interactions between 46 transcription factors and ∼550 candidate target genes comprising 20% of its genome. We predicted ∼170 novel interactions and 24 novel regulons for the control of the central metabolic pathways in
S. aureus
. The reconstructed regulons are largely variable in the
Staphylococcaceae
: only 20% of
S. aureus
regulatory interactions are conserved across all studied genomes. We used a large-scale gene expression data set for
S. aureus
to assess relationships between the inferred regulons and gene expression patterns. The predicted reference set of regulons is captured within the
Staphylococcus
collection in the RegPrecise database (
http://regprecise.lbl.gov
).
Applied Genomics: Data Mining Reveals Species-Specific Malaria Diagnostic Targets More Sensitive than 18S rRNA
Authors: Demas Allison , Oberstaller Jenna , DeBarry Jeremy , Lucchi Naomi W. , Srinivasamoorthy Ganesh , Sumari Deborah , Kabanywanyi Abdunoor M. , Villegas Leopoldo , Escalante Ananias A. , Kachur S. Patrick , Barnwell John W. , Peterson David S. , Udhayakumar Venkatachalam , Kissinger Jessica C.
Published: April 2011

Accurate and rapid diagnosis of malaria infections is crucial for implementing species-appropriate treatment and saving lives. Molecular diagnostic tools are the most accurate and sensitive method of detecting
Plasmodium
, differentiating between
Plasmodium
species, and detecting subclinical infections. Despite available whole-genome sequence data for
Plasmodium falciparum
and
P. vivax
, the majority of PCR-based methods still rely on the 18S rRNA gene targets. Historically, this gene has served as the best target for diagnostic assays. However, it is limited in its ability to detect mixed infections in multiplex assay platforms without the use of nested PCR. New diagnostic targets are needed. Ideal targets will be species specific, highly sensitive, and amenable to both single-step and multiplex PCRs. We have mined the genomes of
P. falciparum
and
P. vivax
to identify species-specific, repetitive sequences that serve as new PCR targets for the detection of malaria. We show that these targets (Pvr47 and Pfr364) exist in 14 to 41 copies and are more sensitive than 18S rRNA when utilized in a single-step PCR. Parasites are routinely detected at levels of 1 to 10 parasites/μl. The reaction can be multiplexed to detect both species in a single reaction. We have examined 7
P. falciparum
strains and 91
P. falciparum
clinical isolates from Tanzania and 10
P. vivax
strains and 96
P. vivax
clinical isolates from Venezuela, and we have verified a sensitivity and specificity of ∼100% for both targets compared with a nested 18S rRNA approach. We show that bioinformatics approaches can be successfully applied to identify novel diagnostic targets and improve molecular methods for pathogen detection. These novel targets provide a powerful alternative molecular diagnostic method for the detection of
P. falciparum
and
P. vivax
in conventional or multiplex PCR platforms.
Natural Substrate Concentrations Can Modulate the Prophylactic Efficacy of Nucleotide HIV Reverse Transcriptase Inhibitors
Authors: García-Lerma J. Gerardo , Aung Wutyi , Cong Mian-er , Zheng Qi , Youngpairoj Ae S. , Mitchell James , Holder Angela , Martin Amy , Kuklenyik Susan , Luo Wei , Lin Carol Yen-Chin , Hanson Debra L. , Kersh Ellen , Pau Chou-Pong , Ray Adrian S. , Rooney James F. , Lee William A. , Heneine Walid
Published: April 2011

Preexposure prophylaxis (PrEP) with antiretroviral drugs is a novel human immunodeficiency virus (HIV) prevention strategy. It is generally thought that high systemic and mucosal drug levels are sufficient for protection. We investigated whether GS7340, a next-generation tenofovir (TFV) prodrug that effectively delivers tenofovir diphosphate (TFV-DP) to lymphoid cells and tissues, could protect macaques against repeated weekly rectal simian-human immunodeficiency virus (SHIV) exposures. Macaques received prophylactic GS7340 treatment 3 days prior to each virus exposure. At 3 days postdosing, TFV-DP concentrations in peripheral blood mononuclear cells (PBMCs) were about 50-fold higher than those seen with TFV disoproxil fumarate (TDF), and they remained above 1,000 fmol/10
6
cells for as long as 7 days. TFV-DP accumulated in lymphoid and rectal tissues, with concentrations at 3 days exceeding 500 fmol/10
6
mononuclear cells. Despite high mucosal and systemic TFV levels, GS7340 was not protective. Since TFV-DP blocks reverse transcription by competing with the natural dATP substrate, we measured dATP contents in peripheral lymphocytes, lymphoid tissue, and rectal mononuclear cells. Compared to those in circulating lymphocytes and lymphoid tissue, rectal lymphocytes had 100-fold higher dATP concentrations and dATP/TFV-DP ratios, likely reflecting the activated status of the cells and suggesting that TFV-DP may be less active at the rectal mucosa. Our results identify dATP/TFV-DP ratios as a possible correlate of protection by TFV and suggest that natural substrate concentrations at the mucosa will likely modulate the prophylactic efficacy of nucleotide reverse transcriptase inhibitors.
Safety and efficacy of intracranial stenting for acute ischemic stroke beyond 8 h of symptom onset
Authors: Xavier Andrew R , Tiwari Ambooj , Purai Natasha , Rayes Mahmoud , Pandey Paritosh , Kansara Amit , Narayanan Sandra , Chaturvedi Seemant
Published: April 2011

Objective

To report our experience with stent supported intracranial recanalization for acute ischemic stroke beyond 8 h of symptoms onset.

Background

Acute ischemic stroke (AIS) therapy is often limited to an 8 h window using mechanical means. However, recent reports have shown delayed recanalization beyond 8 h might be a viable option in a subset of patients.

Methods

A retrospective review was performed of our AIS database for patients who underwent stent supported intracranial recanalization beyond 8 h of symptom onset. Clinical and angiographic data were reviewed. Outcome was measured using modified Rankin Scale (mRS) scores at 30 and 90 days.

Results

12 patients (11 men and one woman) underwent delayed stenting for AIS. Mean age was 49 years (range 37–73) and mean National Institutes of Health Stroke Scale was 17 (range 8–29, median 15). Mean time from stroke onset to intervention was 66.1 h (range 10–168 h, median 46 h). 10 patients presented with a Thrombolysis in Myocardial Infarction (TIMI) score of 0 and the remaining two had a TIMI of 1. Recanalized vessels included: left middle cerebral artery (n=6), basilar trunk (n=2), vertebrobasilar junction (n=3) and internal carotid artery (ICA)-T (n=1). Four patients had prior attempts of embolectomy/thrombolysis using mechanical and chemical means. Stents used included: six balloon mounted stents, five Wingspan and one Enterprise self-expanding intracranial stent. Recanalization, defined as a TIMI score of 2 or more, was achieved in 11 patients. Two patients (17%) had intracranial hemorrhage. Thirty day mRS of ≤3 was achieved in six patients (50%). Seven patients (58%) had a 90 day mRS of ≤2.

Conclusion

Stent supported intracranial recanalization is a safe and feasible approach in a selective group of patients presenting with acute ischemic stroke beyond 8 h of symptom onset.
A BRACKET POLYNOMIAL FOR GRAPHS, III: VERTEX WEIGHTS
Authors: TRALDI LORENZO
Published: April 2011

In earlier work the Kauffman bracket polynomial was extended to an invariant of marked graphs, i.e. looped graphs whose vertices have been partitioned into two classes (marked and not marked). The marked-graph bracket polynomial is readily modified to handle graphs with weighted vertices. We present formulas that simplify the computation of this weighted bracket for graphs that contain twin vertices or are constructed using graph composition, and we show that graph composition corresponds to the construction of a link diagram from tangles.
THELACKOF SEA TO SUPPORT AGRI-ENVIRONMENTAL OBJECTIVES IN IRELAND'S RURAL ENVIRONMENT PROTECTION SCHEME
Authors: WHELAN JACKIE , FRY JOHN
Published: April 2011

Proposals specifically aimed at environmental benefits or enhancements are often exempt from environmental assessment, despite evidence that they can be counter-productive. This is true of agri-environmental schemes where local farm-scale actions are expected to generate large-area cumulative effects on water quality, biodiversity or landscape. Evidence suggests benefits of schemes have often been assumed rather than planned for, necessitating ex-post assessment to justify continuance. This paper argues the need for the Irish Rural Environmental Protection Scheme (REPS) to be assessed using SEA. REPS promoted a range of small-scale improvements in a bottom-up approach intended to reflect several high-level policy objectives primarily concerning environmental protection. The paper is based on analysis of "landscape protection" under the REPS from inception in 1994 until its phasing out period in 2015 when all participants will have completed their plans. The paper investigates how linking SEA to REPS had potential for harmonising with other policy objectives and therefore avoiding administrative duplication. Comparison with the Water Framework Directive suggests opportunities to connect different environmental procedures.
MR-GUIDED PULSE OXIMETRY IMAGING OF BREAST IN VIVO
Authors: LI ZHIQIU , JIANG SHUDONG , KRISHNASWAMY VENKATARAMANAN , DAVIS SCOTT C. , SRINIVASAN SUBHADRA , PAULSEN KEITH D. , POGUE BRIAN W.
Published: April 2011

A near-infrared (NIR) tomography system with spectrally-encoded sources in two wavelength bands was built to quantify the temporal oxyhemoglobin and deoxyhemoglobin contrast in breast tissue at a 20 Hz bandwidth. The system was integrated into a 3 T magnetic resonance (MR) imaging system through a customized breast coil interface for simultaneous optical and MRI acquisition. In this configuration, the MR images provide breast tissue structural information for NIR spectroscopy of adipose and fibro-glandular tissue in breast. Spectral characterization performance of the NIR system was verified through dynamic phantom experiments. Normal human subjects were imaged with finger pulse oximeter (PO) plethysmogram synchronized to the NIR system to provide a frequency-locked reference. Both the raw data from the NIR system and the recovered absorption coefficients of the breast at two wavelengths showed the same frequency of about 1.3 Hz as the PO output. The frequency lock-in approach provided a practical platform for MR-localized recovery of small pulsatile variations of oxyhemoglobin and deoxyhemoglobin in the breast, which are related to the heartbeat and vascular resistance of the tissue.
Hypoxia-induced inhibition of lung development is attenuated by the peroxisome proliferator-activated receptor-γ agonist rosiglitazone
Authors: Nicola Teodora , Ambalavanan Namasivayam , Zhang Wei , James Masheika L. , Rehan Virender , Halloran Brian , Olave Nelida , Bulger Arlene , Oparil Suzanne , Chen Yiu-Fai
Published: April 2011

Hypoxia enhances transforming growth factor-β (TGF-β) signaling, inhibiting alveolar development and causing abnormal pulmonary arterial remodeling in the newborn lung. We hypothesized that, during chronic hypoxia, reduced peroxisome proliferator-activated receptor-γ (PPAR-γ) signaling may contribute to, or be caused by, excessive TGF-β signaling. To determine whether PPAR-γ was reduced during hypoxia, C57BL/6 mice were exposed to hypoxia from birth to 2 wk and evaluated for PPAR-γ mRNA and protein. To determine whether rosiglitazone (RGZ, a PPAR-γ agonist) supplementation attenuated the effects of hypoxia, mice were exposed to air or hypoxia from birth to 2 wk in combination with either RGZ or vehicle, and measurements of lung histology, function, parameters related to TGF-β signaling, and collagen content were made. To determine whether excessive TGF-β signaling reduced PPAR-γ, mice were exposed to air or hypoxia from birth to 2 wk in combination with either TGF-β-neutralizing antibody or vehicle, and PPAR-γ signaling was evaluated. We observed that hypoxia reduced PPAR-γ mRNA and protein, in association with impaired alveolarization, increased TGF-β signaling, reduced lung compliance, and increased collagen. RGZ increased PPAR-γ signaling, with improved lung development and compliance in association with reduced collagen and TGF-β signaling. However, no reduction was noted in hypoxia-induced pulmonary vascular remodeling. Inhibition of hypoxia-enhanced TGF-β signaling increased PPAR-γ signaling. These results suggest that hypoxia-induced inhibition of lung development is associated with a mutually antagonistic relationship between reduced PPAR-γ and increased TGF-β signaling. PPAR-γ agonists may be of potential therapeutic significance in attenuating TGF-β signaling and improving alveolar development.
Mucociliary interactions and mucus dynamics in ciliated human bronchial epithelial cell cultures
Authors: Sears Patrick R. , Davis C. William , Chua Michael , Sheehan John K.
Published: April 2011

The airway epithelial surface liquid is generally considered to be composed of two layers, a periciliary layer and a continuous thick mucus layer moving in bulk. This view may not be appropriate for all areas of the lung. Our hypothesis, that mucus may form a discontinuous layer with dynamic attachments to the surface, is investigated using a culture system. We used live-cell confocal microscopy to investigate thin mucus layers and fluorescent beads and exogenous MUC5B to visualize mucus dynamics on ciliated human bronchial cultures. A continuous mucus layer was not observed. In sparsely ciliated cultures, mucus attached to ciliated cells; however, in highly ciliated cultures, mucus formed strands several hundred micrometers long. As with increases in ciliation, increases in bead concentration caused the appearance of mucus strands. We confirmed the involvement of mucins in the binding of mucus to cilia by adding labeled purified MUC5B to the cultures. These data suggest that mucins may have an intrinsic ability to form attachments to cilia. The significance of these findings is that aberrant modulation of such an intrinsic property may explain the initiation of highly adherent mucus in cystic fibrosis lung disease.
A novel instrumented multipeg running wheel system, Step-Wheel, for monitoring and controlling complex sequential stepping in mice
Authors: Kitsukawa Takashi , Nagata Masatoshi , Yanagihara Dai , Tomioka Ryohei , Utsumi Hideko , Kubota Yasuo , Yagi Takeshi , Graybiel Ann M. , Yamamori Tetsuo
Published: April 2011

Motor control is critical in daily life as well as in artistic and athletic performance and thus is the subject of intense interest in neuroscience. Mouse models of movement disorders have proven valuable for many aspects of investigation, but adequate methods for analyzing complex motor control in mouse models have not been fully established. Here, we report the development of a novel running-wheel system that can be used to evoke simple and complex stepping patterns in mice. The stepping patterns are controlled by spatially organized pegs, which serve as footholds that can be arranged in adjustable, ladder-like configurations. The mice run as they drink water from a spout, providing reward, while the wheel turns at a constant speed. The stepping patterns of the mice can thus be controlled not only spatially, but also temporally. A voltage sensor to detect paw touches is attached to each peg, allowing precise registration of footfalls. We show that this device can be used to analyze patterns of complex motor coordination in mice. We further demonstrate that it is possible to measure patterns of neural activity with chronically implanted tetrodes as the mice engage in vigorous running bouts. We suggest that this instrumented multipeg running wheel (which we name the Step-Wheel System) can serve as an important tool in analyzing motor control and motor learning in mice.
Day and Night GSH and MDA Levels in Healthy Adults and Effects of Different Doses of Melatonin on These Parameters
Authors: Chakravarty Shilpa , Rizvi Syed Ibrahim
Published: April 2011

The pineal secretory product melatonin (chemically, N-acetyl-5-methoxytryptamine) acts as an effective antioxidant and free-radical scavenger and plays an important role in several physiological functions such as sleep induction, immunomodulation, cardiovascular protection, thermoregulation, neuroprotection, tumor-suppression and oncostasis. Membrane lipid-peroxidation in terms of malondialdehyde (MDA) and intracellular glutathione (GSH) is considered to be a reliable marker of oxidative stress. The present work was undertaken to study the modulating effect of melatonin on MDA and GSH in human erythrocytes during day and night. Our observation shows the modulation of these two biomarkers by melatonin, and this may have important therapeutic implications.In vitrodose-dependent effect of melatonin also showed variation during day and night. We explain our observations on the basis of melatonin's antioxidative function and its effect on the fluidity of plasma membrane of red blood cells. Rhythmic modulation of MDA and GSH contents emphasized the role of melatonin as an antioxidant and its function against oxidative stress.
Immunolymphoscintigraphy for Metastatic Sentinel Nodes: Test of a Model
Authors: Chakera A. H. , Nielsen B. S. , Madsen J. , Romer J. , Kristjansen P. E. G. , Buch I. , Binderup T. , Ingvar C. , Nalla A. , Kjaer A. , Hesse B.
Published: April 2011

Aim. To develop a method and obtain proof-of-principle for immunolymphoscintigraphy for identification of metastatic sentinel nodes. Methods. We selected one of four tumour-specific antibodies against human breast cancer and investigated (1), in immune-deficient (nude) mice with xenograft human breast cancer expressing the antigen if specific binding of the intratumorally injected, radioactively labelled, monoclonal antibody could be scintigraphically visualized, and (2) transportation to and retention in regional lymph nodes of the radioactively labelled antibody after subcutaneous injection in healthy rabbits. Results and Conclusion. Our paper suggests the theoretical possibility of a model of dual isotope immuno-lymphoscintigraphy for noninvasive, preoperative, malignant sentinel node imaging.
Absence of MMP2 Expression Correlates with Poor Clinical Outcomes in Rectal Cancer, and Is Distinct from MMP1-Related Outcomes in Colon Cancer
Authors: Wong John C.T. , Chan Simon K. , Schaeffer David F. , Sagaert Xavier , Lim Howard J. , Kennecke Hagen , Owen David A. , Suh Kwang W. , Kim Young-Bae , Tai Isabella T.
Published: April 2011

Purpose: Treatments for colorectal cancer (CRC) are primarily disease stage based. However, heterogeneity in outcome within even a single stage highlights its limitations in predicting disease behavior. Recently, the role of gene expression as predictive and prognostic markers has been explored. Our objectives were to identify consistently differentially expressed genes through meta-analysis of high-throughput gene-expression studies, and evaluate their predictive and prognostic significance in colon (CC) and rectal (RC) cancers.

Experimental Design: Publications applying high-throughput gene- expression technologies to specific CRC stages were identified. A vote counting strategy was used to identify the most significant differentially expressed genes. Their predictive and prognostic values were independently assessed in a tissue microarray of 191 cases of stage II-IV CC/RC from two tertiary care centers. Their biological effects were also examined in vitro.

Results: MMP1 and MMP2 were identified as consistently underexpressed in liver metastasis compared with primary CRC. Shorter time to distant metastasis and overall survival occurred in stage III CC lacking MMP1 expression, and in stage III RC lacking MMP2. MMP1 levels in stage II and III CC were associated with increased likelihood of distant metastasis, whereas the risk of local recurrence in stage III RC could be stratified by MMP2. Promotion of cell invasion of CRC cell lines exposed to MMP1/2 inhibitors were confirmed in vitro.

Conclusions: MMP1 and MMP2 may be useful biomarkers that can help stratify patients at higher risk of developing recurrence in colorectal cancer, and guide individualized treatment decisions to achieve better outcomes. Clin Cancer Res; 17(12); 4167–76. ©2011 AACR.
Changes in Tumor Density in Patients with Advanced Hepatocellular Carcinoma Treated with Sunitinib
Authors: Faivre Sandrine , Zappa Magaly , Vilgrain Valérie , Boucher Eveline , Douillard Jean-Yves , Lim Ho Y. , Kim Jun S. , Im Seock-Ah , Kang Yoon-Koo , Bouattour Mohamed , Dokmak Safi , Dreyer Chantal , Sablin Marie-Paule , Serrate Camille , Cheng Ann-Lii , Lanzalone Silvana , Lin Xun , Lechuga Maria J. , Raymond Eric
Published: April 2011

Purpose: Response Evaluation Criteria in Solid Tumors (RECIST) may underestimate the efficacy of targeted therapies. In hepatocellular carcinoma (HCC) studies with sunitinib, RECIST-defined response rates are low, although hypodensity on computed tomography (CT) scans occurs more frequently. This exploratory analysis investigated tumor density as a surrogate endpoint of sunitinib activity in a phase II HCC study.

Experimental Design: Patients received sunitinib 50 mg/d (4 weeks on/2 weeks off). Tumor size and density were assessed on CT scans by using RECIST and Choi criteria, the latter of which classify a partial response as a 15% or more reduction in tumor density or a 10% or more reduction in tumor size. The overall percentage volume of tumor necrosis was calculated with volumetric reconstruction. Tumor perfusion parameters were assessed by using perfusion CT scans with specific acquisition.

Results: Among the 26 evaluable patients, 1 achieved a partial response and 22 had tumor stabilization by RECIST. In analysis of tumor density, 17 of 26 patients (65.4%) were responders by Choi criteria. Volumetric assessment showed major tumor necrosis (≥30% of tumor volume) in 10 of 21 patients (47.6%). Among four patients evaluated, tumor blood flow was reduced by 58.8% and blood volume by 68.4% after 4 weeks of treatment. The median time to progression (TTP) was 6.4 months. Patients with responses by Choi criteria had a significantly longer TTP (7.5 months) compared with nonresponders (4.8 months; HR = 0.33, two-sided P = 0.0182).

Conclusions: Tumor density assessment suggested that radiologic endpoints in addition to RECIST may be considered to capture sunitinib activity in HCC. Clin Cancer Res; 17(13); 4504–12. ©2011 AACR.
Dopamine Blocks Stress-Mediated Ovarian Carcinoma Growth
Authors: Moreno-Smith Myrthala , Lu Chunhua , Shahzad Mian M.K. , Pena Guillermo N. Armaiz , Allen Julie K. , Stone Rebecca L. , Mangala Lingegowda S. , Han Hee Dong , Kim Hye Sun , Farley Donna , Berestein Gabriel Lopez , Cole Steve W. , Lutgendorf Susan K. , Sood Anil K.
Published: April 2011

Purpose: Increased adrenergic activity in response to chronic stress is known to promote tumor growth by stimulating the tumor microenvironment. The focus of the current study was to determine whether dopamine, an inhibitory catecholamine, could block the effects of chronic stress on tumor growth.

Experimental Design: Expression of dopamine receptors (DR1–DR5) was analyzed by reverse transcriptase-PCR and by Western blotting. In vitro effects of dopamine on cell viability, apoptosis, and migration were examined. For in vivo therapy, murine and human DR2-siRNAs were incorporated into chitosan nanoparticles (CH-NP).

Results: In this model of chronic stress, tumoral norepinephrine levels remained elevated whereas dopamine levels were significantly decreased compared with nonstressed animals. Daily restraint stress resulted in significantly increased tumor growth in both immunodeficient (SKOV3ip1 and HeyA8) and immunocompetent (ID8) ovarian cancer models. This increase was completely blocked with daily dopamine treatment. Dopamine treatment also blocked the stress-induced increase in angiogenesis. Endothelial and ovarian cancer cells expressed all dopamine receptors except for the lack of DR3 expression in ovarian cancer cells. DR2 was responsible for the inhibitory effects of dopamine on tumor growth and microvessel density as well as the stimulatory effect on apoptosis, as the DR2 antagonist eticlopride reversed these effects. Dopamine significantly inhibited cell viability and stimulated apoptosis in vitro. Moreover, dopamine reduced cyclic AMP levels and inhibited norepinephrine and vascular permeability factor/VEGF-induced Src kinase activation.

Conclusions: Dopamine depletion under chronic stress conditions creates a permissive microenvironment for tumor growth that can be reversed by dopamine replacement. Clin Cancer Res; 17(11); 3649–59. ©2011 AACR.
Prospective Gene Signature Study Using microRNA to Identify the Tissue of Origin in Patients with Carcinoma of Unknown Primary
Authors: Varadhachary Gauri R. , Spector Yael , Abbruzzese James L. , Rosenwald Shai , Wang Huamin , Aharonov Ranit , Carlson Heather R. , Cohen Dalia , Karanth Siddharth , Macinskas Joanna , Lenzi Renato , Chajut Ayelet , Edmonston Tina B. , Raber Martin N.
Published: April 2011

Purpose: Accurate identification of tissue of origin (ToO) for patients with carcinoma of unknown primary (CUP) may help customize therapy to the putative primary and thereby improve the clinical outcome. We prospectively studied the performance of a microRNA-based assay to identify the ToO in CUP patients.

Experimental Design: Formalin-fixed paraffin-embedded (FFPE) metastatic tissue from 104 patients was reviewed and 87 of these contained sufficient tumor for testing. The assay quantitates 48 microRNAs and assigns one of 25 tumor diagnoses by using a biologically motivated binary decision tree and a K-nearest neighbors (KNN). The assay predictions were compared with clinicopathologic features and, where suitable, to therapeutic response.

Results: Seventy-four of the 87 cases were processed successfully. The assay result was consistent or compatible with the clinicopathologic features in 84% of cases processed successfully (71% of all samples attempted). In 65 patients, pathology and immunohistochemistry (IHC) suggested a diagnosis or (more often) a differential diagnosis. Out of those, the assay was consistent or compatible with the clinicopathologic presentation in 55 (85%) cases. Of the 9 patients with noncontributory IHC, the assay provided a ToO prediction that was compatible with the clinical presentation in 7 cases.

Conclusions: In this prospective study, the microRNA diagnosis was compatible with the clinicopathologic picture in the majority of cases. Comparative effectiveness research trials evaluating the added benefit of molecular profiling in appropriate CUP subsets are warranted. MicroRNA profiling may be particularly helpful in patients in whom the IHC profile of the metastasis is nondiagnostic or leaves a large differential diagnosis. Clin Cancer Res; 17(12); 4063–70. ©2011 AACR.
A Low-Toxicity IL-2–Based Immunocytokine Retains Antitumor Activity Despite Its High Degree of IL-2 Receptor Selectivity
Authors: Gillies Stephen D. , Lan Yan , Hettmann Thore , Brunkhorst Beatrice , Sun Yaping , Mueller Stefan O. , Lo Kin-Ming
Published: April 2011

Purpose: The goal of the study was to engineer a form of interleukin 2 (IL-2) that, when delivered as a tumor-specific antibody fusion protein, retains the ability to stimulate an antitumor immune response via interaction with the high-affinity IL-2 receptor but has lower toxicity because of the reduced activation of the intermediate-affinity IL-2 receptor.

Experimental Design: We investigated changes in the proposed toxin motif of IL-2 by introducing a D20T mutation that has little effect on the activity of free IL-2. We expressed this IL-2 variant as a fusion protein with an antibody (NHS76) that targets the necrotic core of tumors and characterized this molecule (NHS-IL2LT) in vitro and in vivo.

Results: NHS-IL2LT was shown to have near normal biological activity in vitro by using T-cell lines expressing the high-affinity IL-2 receptor, but little or no activity by using cell lines expressing only the intermediate IL-2 receptor. Relative to the control antibody fusion protein containing wild-type IL-2, NHS-IL2LT retained antitumor activity against established neuroblastoma and non–small cell lung cancer metastases in syngeneic mouse tumor models but was much better tolerated in immune-competent mice and in cynomolgus monkeys.

Conclusions: The qualities of low toxicity and single-agent efficacy shown suggest that NHS-IL2LT is a good candidate for therapeutic approaches combining standard cytotoxic and immune therapies. In fact, this molecule (also known as Selectikine or EMD 521873) is currently in phase I clinical trial. Clin Cancer Res; 17(11); 3673–85. ©2011 AACR.