bluuebunny/crossref_metadata_2025 huggingface dataset:
The Nitrogenase Regulatory Enzyme Dinitrogenase Reductase ADP-Ribosyltransferase (DraT) Is Activated by Direct Interaction with the Signal Transduction Protein GlnB
Authors: Moure Vivian R. , Danyal Karamatullah , Yang Zhi-Yong , Wendroth Shannon , Müller-Santos Marcelo , Pedrosa Fabio O. , Scarduelli Marcelo , Gerhardt Edileusa C. M. , Huergo Luciano F. , Souza Emanuel M. , Seefeldt Lance C.
Published: November 2012

Fe protein (dinitrogenase reductase) activity is reversibly inactivated by dinitrogenase reductase ADP-ribosyltransferase (DraT) in response to an increase in the ammonium concentration or a decrease in cellular energy inAzospirillum brasilense,Rhodospirillum rubrum, andRhodobacter capsulatus. The ADP-ribosyl is removed by the dinitrogenase reductase-activating glycohydrolase (DraG), promoting Fe protein reactivation. The signaling pathway leading to DraT activation by ammonium is still not completely understood, but the available evidence shows the involvement of direct interaction between the enzyme and the nitrogen-signaling PIIproteins. InA. brasilense, two PIIproteins, GlnB and GlnZ, were identified. We used Fe protein fromAzotobacter vinelandiias the substrate to assess the activity ofA. brasilenseDraTin vitrocomplexed or not with PIIproteins. Under our conditions, GlnB was necessary for DraT activity in the presence of Mg-ADP. The PIIeffector 2-oxoglutarate, in the presence of Mg-ATP, inhibited DraT-GlnB activity, possibly by inducing complex dissociation. DraT was also activated by GlnZ and by both uridylylated PIIproteins, but not by a GlnB variant carrying a partial deletion of the T loop. Kinetics studies revealed that theA. brasilenseDraT-GlnB complex was at least 18-fold more efficient than DraT purified fromR. rubrum, but with a similarKmvalue for NAD+. Our results showed that ADP-ribosylation of the Fe protein does not affect the electronic state of its metal cluster and prevents association between the Fe and MoFe proteins, thus inhibiting electron transfer.
Flagellum Density Regulates Proteus mirabilis Swarmer Cell Motility in Viscous Environments
Authors: Tuson Hannah H. , Copeland Matthew F. , Carey Sonia , Sacotte Ryan , Weibel Douglas B.
Published: November 2012

Proteus mirabilis
is an opportunistic pathogen that is frequently associated with urinary tract infections. In the lab,
P. mirabilis
cells become long and multinucleate and increase their number of flagella as they colonize agar surfaces during swarming. Swarming has been implicated in pathogenesis; however, it is unclear how energetically costly changes in
P. mirabilis
cell morphology translate into an advantage for adapting to environmental changes. We investigated two morphological changes that occur during swarming—increases in cell length and flagellum density—and discovered that an increase in the surface density of flagella enabled cells to translate rapidly through fluids of increasing viscosity; in contrast, cell length had a small effect on motility. We found that swarm cells had a surface density of flagella that was ∼5 times larger than that of vegetative cells and were motile in fluids with a viscosity that inhibits vegetative cell motility. To test the relationship between flagellum density and velocity, we overexpressed FlhD
4
C
2
, the master regulator of the flagellar operon, in vegetative cells of
P. mirabilis
and found that increased flagellum density produced an increase in cell velocity. Our results establish a relationship between
P. mirabilis
flagellum density and cell motility in viscous environments that may be relevant to its adaptation during the infection of mammalian urinary tracts and movement in contact with indwelling catheters.
FliW and FliS Function Independently To Control Cytoplasmic Flagellin Levels in Bacillus subtilis
Authors: Mukherjee Sampriti , Babitzke Paul , Kearns Daniel B.
Published: November 2012

The cytoplasmic level of flagellin (called Hag) is homeostatically regulated in the Gram-positive bacterium
Bacillus subtilis
by a partner-switching mechanism between the protein FliW and either the Hag structural protein or CsrA, an RNA binding protein that represses
hag
translation. Here we show that FliW and the putative secretion chaperone FliS bind to Hag simultaneously but control Hag translation by different mechanisms. While FliW directly inhibits CsrA activity, FliS antagonizes CsrA indirectly by binding to Hag, enhancing Hag secretion, and depleting Hag in the cytoplasm to trigger the FliW partner switch. Consistent with a role for FliS in potentiating Hag secretion, the mutation of
fliS
crippled both motility and flagellar filament assembly, and both phenotypes could be partially rescued by artificially increasing the concentration of the Hag substrate through the absence of CsrA. Furthermore, the absence of FliS resulted in an approximately 30-fold reduction in extracellular Hag accumulation in cells mutated for CsrA (to relieve homeostatic control) and the filament cap protein FliD (to secrete flagellin into the supernatant). Thus, we mechanistically discriminate between the FliW regulator and the FliS chaperone to show that secretion disrupts flagellin homeostasis and promotes high-level flagellin synthesis during the period of filament assembly in
B. subtilis
.
High-Mobility-Group A-Like CarD Binds to a DNA Site Optimized for Affinity and Position and to RNA Polymerase To Regulate a Light-Inducible Promoter in Myxococcus xanthus
Authors: García-Heras Francisco , Abellón-Ruiz Javier , Murillo Francisco J. , Padmanabhan S. , Elías-Arnanz Montserrat
Published: November 2012

The CarD-CarG complex controls various cellular processes in the bacteriumMyxococcus xanthusincluding fruiting body development and light-induced carotenogenesis. The CarD N-terminal domain, which defines the large CarD_CdnL_TRCF protein family, binds to CarG, a zinc-associated protein that does not bind DNA. The CarD C-terminal domain resembles eukaryotic high-mobility-group A (HMGA) proteins, and its DNA binding AT hooks specifically recognize the minor groove of appropriately spaced AT-rich tracts. Here, we investigate the determinants of the only known CarD binding site, the one crucial in CarD-CarG regulation of the promoter of thecarQRSoperon (PQRS), a light-inducible promoter dependent on the extracytoplasmic function (ECF) σ factor CarQ.In vitro, mutating either of the 3-bp AT tracts of this CarD recognition site (TTTCCAGAGCTTT) impaired DNA binding, shifting the AT tracts relative to PQRShad no effect or marginally lowered DNA binding, and replacing the native site by the HMGA1a binding one at the human beta interferon promoter (with longer AT tracts) markedly enhanced DNA binding.In vivo, however, all of these changes deterred PQRSactivation in wild-typeM. xanthus, as well as in a strain with the CarD-CarG pair replaced by theAnaeromyxobacter dehalogenansCarD-CarG (CarDAd-CarGAd). CarDAd-CarGAdis functionally equivalent to CarD-CarG despite the lower DNA binding affinityin vitroof CarDAd, whose C-terminal domain resembles histone H1 rather than HMGA. We show that CarD physically associates with RNA polymerase (RNAP) specifically via interactions with the RNAP β subunit. Our findings suggest that CarD regulates a light-inducible, ECF σ-dependent promoter by coupling RNAP recruitment and binding to a specific DNA site optimized for affinity and position.
Cloning, Expression, and Functional Characterization of Secondary Amino Acid Transporters of Lactococcus lactis
Authors: Trip Hein , Mulder Niels L. , Lolkema Juke S.
Published: November 2012

Fourteen genes encoding putative secondary amino acid transporters were identified in the genomes of
Lactococcus lactis
subsp.
cremoris
strains MG1363 and SK11 and
L. lactis
subsp
. lactis
strains IL1403 and KF147, 12 of which were common to all four strains. Amino acid uptake in
L. lactis
cells overexpressing the genes revealed transporters specific for histidine, lysine, arginine, agmatine, putrescine, aromatic amino acids, acidic amino acids, serine, and branched-chain amino acids. Substrate specificities were demonstrated by inhibition profiles determined in the presence of excesses of the other amino acids. Four knockout mutants, lacking the lysine transporter LysP, the histidine transporter HisP (formerly LysQ), the acidic amino acid transporter AcaP (YlcA), or the aromatic amino acid transporter FywP (YsjA), were constructed. The LysP, HisP, and FywP deletion mutants showed drastically decreased rates of uptake of the corresponding substrates at low concentrations. The same was observed for the AcaP mutant with aspartate but not with glutamate. In rich M17 medium, the deletion of none of the transporters affected growth. In contrast, the deletion of the HisP, AcaP, and FywP transporters did affect growth in a defined medium with free amino acids as the sole amino acid source. HisP was essential at low histidine concentrations, and AcaP was essential in the absence of glutamine. FywP appeared to play a role in retaining intracellularly synthesized aromatic amino acids when these were not added to the medium. Finally, HisP, AcaP, and FywP did not play a role in the excretion of accumulated histidine, glutamate, or phenylalanine, respectively, indicating the involvement of other transporters.
The Francisella tularensis FabI Enoyl-Acyl Carrier Protein Reductase Gene Is Essential to Bacterial Viability and Is Expressed during Infection
Authors: Kingry Luke C. , Cummings Jason E. , Brookman Kerry W. , Bommineni Gopal R. , Tonge Peter J. , Slayden Richard A.
Published: November 2012

Francisella tularensis
is classified as a category A priority pathogen and causes fatal disseminated disease in humans upon inhalation of less than 50 bacteria. Although drugs are available for treatment, they are not ideal because of toxicity and route of delivery, and in some cases patients relapse upon withdrawal. We have an ongoing program to develop novel FAS-II FabI enoyl-ACP reductase enzyme inhibitors for
Francisella
and other select agents. To establish
F. tularensis
FabI (FtFabI) as a clinically relevant drug target, we demonstrated that fatty acid biosynthesis and FabI activity are essential for growth even in the presence of exogenous long-chain lipids and that Ft
fabI
is not transcriptionally altered in the presence of exogenous long-chain lipids. Inhibition of FtFabI or fatty acid synthesis results in loss of viability that is not rescued by exogenous long-chain lipid supplementation. Importantly, whole-genome transcriptional profiling of
F. tularensis
with DNA microarrays from infected tissues revealed that Ft
fabI
and
de novo
fatty acid biosynthetic genes are transcriptionally active during infection. This is the first demonstration that the FabI enoyl-ACP-reductase enzyme encoded by
F. tularensis
is essential and not bypassed by exogenous fatty acids and that
de novo
fatty acid biosynthetic components encoded in
F. tularensis
are transcriptionally active during infection in the mouse model of tularemia.
Detection of Campylobacter Species and Arcobacter butzleri in Stool Samples by Use of Real-Time Multiplex PCR
Authors: de Boer Richard F. , Ott Alewijn , Güren Pınar , van Zanten Evert , van Belkum Alex , Kooistra-Smid Anna M. D.
Published: November 2012

The presence of
Campylobacter
(or
Campylobacter
-like) species in stools from patients suspected of infectious gastroenteritis (
n
= 493) was investigated using real-time PCR for detection of
Arcobacter butzleri
(
hsp60
gene),
Campylobacter coli
(
ceuE
gene),
Campylobacter jejuni
(
mapA
), five acknowledged pathogenic
Campylobacter
spp. (C16S_Lund assay), and the
Campylobacter
genus (C16S_LvI assay). In total, 71.4% of the samples were positive for
Campylobacter
DNA (
n
= 352) by a
Campylobacter
genus-specific (C16S_LvI) assay. A total of 23 samples (4.7%) were positive in the C16S_Lund assay, used for detection of
C. jejuni
,
C. coli
,
C. lari
,
C. upsaliensis
, and
C. hyointestinalis
. Subsequent identification of these samples yielded detection frequencies (DF) of 4.1% (
C. jejuni
), 0.4% (
C. coli
), and 0.4% (
C. upsaliensis
). The DF of
A. butzleri
was 0.4%. Interestingly, sequencing of a subgroup (
n
= 46) of C16S_LvI PCR-positive samples resulted in a considerable number of
Campylobacter concisus
-positive samples (
n
= 20). PCR-positive findings with the C16S_Lund and
C. jejuni
/
C. coli
-specific assays were associated with more serious clinical symptoms (diarrhea and blood). Threshold cycle (

C
T

) values of
C. jejuni
/
C. coli
PCR-positive samples were comparable to those of the C16S_Lund PCR (
P
= 0.21).

C
T

values for both assays were significantly lower than those of the C16S_LvI assay (
P
< 0.001 and
P
< 0.00001, respectively). In conclusion, this study demonstrated that in combination, the
C. jejuni
/
C coli
-specific assays and the C16S_Lund assay are both useful for routine screening purposes. Furthermore, the DF of the emerging pathogen
C. concisus
was at least similar to the DF of
C. jejuni
.
Trimeric Glycosylphosphatidylinositol-Anchored HCDR3 of Broadly Neutralizing Antibody PG16 Is a Potent HIV-1 Entry Inhibitor
Authors: Liu Lihong , Wang Weiming , Yang Lifei , Ren Huanhuan , Kimata Jason T. , Zhou Paul
Published: November 2012

PG9 and PG16 are two quaternary-structure-specific broadly neutralizing antibodies with unique HCDR3 subdomains. Previously, we showed that glycosylphosphatidylinositol (GPI)-anchored HCDR3 subdomains (GPI-HCDR3) can be targeted to lipid rafts of the plasma membrane, bind to the epitope recognized by HCDR3 of PG16, and neutralize diverse HIV-1 isolates. In this study, we further developed trimeric GPI-HCDR3s and demonstrated that trimeric GPI-HCDR3 (PG16) dramatically improves anti-HIV-1 neutralization, suggesting that a stoichiometry of recognition of 3 or 2 HCDR3 molecules (PG16) to 1 viral spike is possible.
Herpes Simplex Virus 2 (HSV-2) Prevents Dendritic Cell Maturation, Induces Apoptosis, and Triggers Release of Proinflammatory Cytokines: Potential Links to HSV-HIV Synergy
Authors: Stefanidou Martha , Ramos Irene , Mas Casullo Veronica , Trépanier Janie B. , Rosenbaum Sara , Fernandez-Sesma Ana , Herold Betsy C.
Published: November 2012

Herpes simplex virus 2 (HSV-2) may cause frequent recurrences, highlighting its ability to evade host defense. This study tested the hypothesis that HSV-2 interferes with dendritic cell (DC) function as an escape mechanism, which may contribute to enhanced HIV replication in coinfected populations. Immature monocyte-derived human DCs were exposed to live or UV-inactivated HSV-2 or lipopolysaccharide. Little or no increase in the maturation marker CD83 was observed in response to HSV-2 and HSV-2 exposed DCs were impaired in their ability to present antigen (influenza) to T cells. Exposure to UV-inactivated virus stimulated a modest, but significant increase in CD83, suggesting that viral gene expression contributes to the block in DC maturation. The functional impairment of HSV-2-exposed DCs could be partially attributed to the induction of apoptosis. Live and inactivated HSV-2 triggered an increase in the number of early and late apoptotic cells in both the infected and bystander cell populations; apoptosis was associated with a decrease in cellular FLICE-inhibitory protein (c-FLIP). Paradoxically, HSV-2 induced Akt phosphorylation, which typically promotes DC maturation and survival. Despite these aberrant responses, live and inactivated HSV-2 induced the release of cytokines into culture supernatants, which were sufficient to activate HIV-1 replication in latently infected U1 cells. Together, these findings suggest that in the presence of overt or subclinical HSV-2, the function of mucosal DCs would be impaired. These responses may allow HSV to escape immune surveillance but may also promote HIV infection and contribute to the epidemiological link between HIV and HSV.
Expansion of Monocytic Myeloid-Derived Suppressor Cells Dampens T Cell Function in HIV-1-Seropositive Individuals
Authors: Qin Aiping , Cai Weiping , Pan Ting , Wu Kang , Yang Qiong , Wang Nina , Liu Yufeng , Yan Dehong , Hu Fengyu , Guo Pengle , Chen Xiaoping , Chen Ling , Zhang Hui , Tang Xiaoping , Zhou Jie
Published: November 2012

T lymphocyte dysfunction contributes to human immunodeficiency virus type 1 (HIV-1) disease progression by impairing antivirus cellular immunity. However, the mechanisms of HIV-1 infection-mediated T cell dysfunction are not completely understood. Here, we provide evidence that expansion of monocytic myeloid-derived suppressor cells (M-MDSCs) suppressed T cell function in HIV-1-infected individuals. We observed a dramatic elevation of M-MDSCs (HLA-DR
−/low
CD11b
+
CD33
+/high
CD14
+
CD15
−
cells) in the peripheral blood of HIV-1-seropositive subjects (
n
= 61) compared with healthy controls (
n
= 51), despite efficacious antiretroviral therapy for nearly 2 years. The elevated M-MDSC frequency in HIV-1
+
subjects correlated with prognostic HIV-1 disease markers, including the HIV-1 load (
r
= 0.5957;
P
< 0.0001), CD4
+
T cell loss (
r
= −0.5312;
P
< 0.0001), and activated T cells (
r
= 0.4421;
P
= 0.0004). Functional studies showed that M-MDSCs from HIV-1
+
subjects suppressed T cell responses in both HIV-1-specific and antigen-nonspecific manners; this effect was dependent on the induction of arginase 1 and required direct cell-cell contact. Further investigations revealed that direct HIV-1 infection or culture with HIV-1-derived Tat protein significantly enhanced human MDSC generation
in vitro
, and MDSCs from healthy donors could be directly infected by HIV-1 to facilitate HIV-1 replication and transmission, indicating that a positive-feedback loop between HIV-1 infection and MDSC expansion existed. In summary, our studies revealed a novel mechanism of T cell dysfunction in HIV-1-infected individuals and suggested that targeting MDSCs may be a promising strategy for HIV-1 immunotherapy.
Acetylation of Conserved Lysines in Bovine Papillomavirus E2 by p300
Authors: Quinlan Edward J. , Culleton Sara P. , Wu Shwu-Yuan , Chiang Cheng-Ming , Androphy Elliot J.
Published: November 2012

The p300, CBP, and pCAF lysine acetyltransferase (KAT) proteins have been reported to physically interact with bovine (BPV) and human (HPV) papillomavirus E2 proteins. While overexpression of these KAT proteins enhances E2-dependent transcription, the mechanism has not been determined. Using RNA interference (RNAi) to deplete these factors, we demonstrated that E2 transcriptional activity requires physiological levels of p300, CBP, and pCAF. Each protein appears to have a unique function in E2-dependent transcription, since overexpression of one KAT failed to compensate for RNAi knockdown of another KAT. Using an
in vitro
acetylation assay, we identified highly conserved lysines that are targeted by p300 for acetylation. The conservative changes of lysines at positions 111 and 112 to arginine were of particular interest. The K111R and the K111R/K112R mutants showed reduced transcriptional activity that was not responsive to p300 overexpression, while the K112R mutant retained activity. p300 and CBP were detected at the viral promoter; however, pCAF was not. We propose a model by which E2 transcriptional activity is controlled by p300-mediated acetylation of lysine 111. This model represents a novel mechanism regulating papillomavirus gene expression.
Novel Mutations in gB and gH Circumvent the Requirement for Known gD Receptors in Herpes Simplex Virus 1 Entry and Cell-to-Cell Spread
Authors: Uchida Hiroaki , Chan Janet , Shrivastava Indira , Reinhart Bonnie , Grandi Paola , Glorioso Joseph C. , Cohen Justus B.
Published: November 2012

Both entry and cell-to-cell spread of herpes simplex virus (HSV) involve a cascade of cooperative interactions among the essential glycoproteins D, B, and H/L (gD, gB, and gH/gL, respectively) initiated by the binding of gD to a cognate HSV entry receptor. We previously reported that a variant (D285N/A549T) of glycoprotein B (gB:NT) enabled primary virus entry into cells that were devoid of typical HSV entry receptors. Here, we compared the activities of the gB:NT variant with those of a newly selected variant of glycoprotein H (gH:KV) and a frequently coselected gB variant (gB:S668N). In combination, gH:KV and gB:S668N enabled primary virus entry into cells that lacked established HSV entry receptors as efficiently as did gB:NT, but separately, each variant enabled only limited entry. Remarkably, gH:KV uniquely facilitated secondary virus spread between cells that lacked canonical entry receptors. Transient expression of the four essential entry glycoproteins revealed that gH:KV, but not gB:NT, induced fusion between cells lacking the standard receptors. Because the involvement of gD remained essential for virus spread and cell fusion, we propose that gH:KV mimics a transition state of gH that responds efficiently to weak signals from gD to reach the active state. Computational modeling of the structures of wild-type gH and gH:KV revealed relatively subtle differences that may have accounted for our experimental findings. Our study shows that (i) the dependence of HSV-1 entry and spread on specific gD receptors can be reduced by sequence changes in the downstream effectors gB and gH, and (ii) the relative roles of gB and gH are different in entry and spread.
Transcriptional Response of Mucoid Pseudomonas aeruginosa to Human Respiratory Mucus
Authors: Cattoir V. , Narasimhan G. , Skurnik D. , Aschard H. , Roux D. , Ramphal R. , Jyot J. , Lory S.
Published: November 2012

Adaptation of bacterial pathogens to a host can lead to the selection and accumulation of specific mutations in their genomes with profound effects on the overall physiology and virulence of the organisms. The opportunistic pathogenPseudomonas aeruginosais capable of colonizing the respiratory tract of individuals with cystic fibrosis (CF), where it undergoes evolution to optimize survival as a persistent chronic human colonizer. The transcriptome of a host-adapted, alginate-overproducing isolate from a CF patient was determined following growth of the bacteria in the presence of human respiratory mucus. This stable mucoid strain responded to a number of regulatory inputs from the mucus, resulting in an unexpected repression of alginate production. Mucus in the medium also induced the production of catalases and additional peroxide-detoxifying enzymes and caused reorganization of pathways of energy generation. A specific antibacterial type VI secretion system was also induced in mucus-grown cells. Finally, a group of small regulatory RNAs was identified and a fraction of these were mucus regulated. This report provides a snapshot of responses in a pathogen adapted to a human host through assimilation of regulatory signals from tissues, optimizing its long-term survival potential.

IMPORTANCEThe basis for chronic colonization of patients with cystic fibrosis (CF) by the opportunistic pathogenPseudomonas aeruginosacontinues to represent a challenging problem for basic scientists and clinicians. In this study, the host-adapted, alginate-overproducingPseudomonas aeruginosa2192 strain was used to assess the changes in its transcript levels following growth in respiratory CF mucus. Several significant and unexpected discoveries were made: (i) although the alginate overproduction in strain 2192 was caused by a stable mutation, a mucus-derived signal caused reduction in the transcript levels of alginate biosynthetic genes; (ii) mucus activated the expression of the type VI secretion system, a mechanism for killing of other bacteria in a mixed population; (iii) expression of a number of genes involved in respiration was altered; and (iv) several small regulatory RNAs were identified, some being mucus regulated. This work highlights the strong influence of the host environment in shaping bacterial survival strategies.
Antibiotic-Mediated Selection of Quorum-Sensing-Negative Staphylococcus aureus
Authors: Paulander Wilhelm , Nissen Varming Anders , Bæk Kristoffer T. , Haaber Jakob , Frees Dorte , Ingmer Hanne
Published: November 2012

Staphylococcus aureus
is a human commensal that at times turns into a serious bacterial pathogen causing life-threatening infections. For the delicate control of virulence,
S. aureus
employs the
agr
quorum-sensing system that, via the intracellular effector molecule RNAIII, regulates virulence gene expression. We demonstrate that the presence of the
agr
locus imposes a fitness cost on
S. aureus
that is mediated by the expression of RNAIII. Further, we show that exposure to sublethal levels of the antibiotics ciprofloxacin, mupirocin, and rifampin, each targeting separate cellular functions, markedly increases the
agr
-mediated fitness cost by inducing the expression of RNAIII. Thus, the extensive use of antibiotics in hospitals may explain why
agr
-negative variants are frequently isolated from hospital-acquired
S. aureus
infections but rarely found among community-acquired
S. aureus
strains. Importantly,
agr
deficiency correlates with increased duration of and mortality due to bacteremia during antibiotic treatment and with a higher frequency of glycopeptide resistance than in
agr
-carrying strains. Our results provide an explanation for the frequent isolation of
agr
-defective strains from hospital-acquired
S. aureus
infections and suggest that the adaptability of
S. aureus
to antibiotics involves the
agr
locus.

IMPORTANCE
Staphylococcus aureus
is the most frequently isolated pathogen in intensive care units and a common cause of nosocomial infections, resulting in a high degree of morbidity and mortality. Surprisingly, a large fraction (15 to 60%) of hospital-isolated
S. aureus
strains are
agr
defective and lack the main quorum-sensing-controlled virulence regulatory system. This is a problem, as
agr
-defective strains are associated with a mortality level in bacteremic infections and a probability of glycopeptide resistance greater than those of other strains. We show here that
agr
-negative strains have a fitness advantage over
agr
-positive strains in the presence of sublethal concentrations of some antibiotics and that the fitness defect of
agr
-positive cells is caused by antibiotic-mediated expression of the
agr
effector molecule RNAIII. These results offer an explanation of the frequent isolation of
agr
-defective
S. aureus
strains in hospitals and will influence how we treat
S. aureus
infections.
A long-lived life history for a tropical, deepwater snapper (Pristipomoides filamentosus): bomb radiocarbon and lead–radium dating as extensions of daily increment analyses in otoliths
Authors: Andrews Allen H. , DeMartini Edward E. , Brodziak Jon , Nichols Ryan S. , Humphreys Robert L.
Published: November 2012

Growth characteristics of Pristipomoides filamentosus, a deepwater eteline snapper of major economic importance, are incomplete and inconsistent across its geographical range. Early growth rates have been validated using daily increment and length–frequency analyses, but historical estimates of adult growth rates are variable and longevity is unknown. Studies of P. filamentosus in the Hawaiian Islands have cautioned against unjustified estimates of longevity, but 18 years has at times been uncritically assumed as the maximum age. The present study addresses these age, growth, and longevity issues using lead–radium and bomb radiocarbon dating by providing valid age estimates for adult P. filamentosus. Valid length-at-age estimates ranged from approximately 10 years to more than 40 years. These data, together with robust daily increment data, were used to model a fully validated, long-lived life history for P. filamentosus. This study adds to the few existing studies supporting a view that many tropical fishes, particularly deepwater species, can be longer lived than previously surmised.
A QUANTITATIVE PROCEDURE FOR THE SPATIAL CHARACTERIZATION OF URBAN LAND USE
Authors: DECRAENE JAMES , MONTEROLA CHRISTOPHER , LEE GARY KEE KHOON , HUNG TERENCE GIH GUANG
Published: November 2012

We have developed a procedure that characterizes the land use pattern of an urban system using: (a) Spatial entropy that measures the extent of spread of residential, business and industrial sectors; and (b) Index of dissimilarity that quantifies the degree of mixing in space of different sectors. The approach is illustrated by using the land use zoning maps of the city state of Singapore and a selection of North American cities. We show that a common feature of most cities is for the industrial areas to be highly clustered while at the same time segregated from the residential or business districts. We also demonstrate that the combination of entropy of residential and dissimilarity index between residential and business areas provides a quantitative and potentially useful means of differentiating the land use pattern of different cities.
AB INITIO STUDY OF OPTOELECTRONIC PROPERTIES OF SPINEL ZnAl2O4 BEYOND GGA AND LDA
Authors: YOUSAF MASOOD , SAEED M. A. , ISA AHMAD RADZI MAT , RAHNAMAYE ALIABAD H. A. , NOOR N. A.
Published: November 2012

Electronic band structure and optical parameters of ZnAl 2 O 4 are investigated by first-principles technique based on a new potential approximation, known as modified Becke–Johnson (mBJ). This method describes the excited states of insulators and semiconductors more accurately The recent direct band gap result by EV-GGA is underestimated by about 15% compared to our band gap value using mBJ-GGA. The value of the band gap of ZnAl 2 O 4 decreases as follows: Eg (mBJ-GGA/LDA) > Eg (GGA) > Eg (LDA) . The band structure base optical parametric quantities (dielectric constant, index of refraction, reflectivity and optical conductivity) are also calculated, and their variations with energy range are discussed. The first critical point (optical absorption's edge) in ZnAl 2 O 4 occurs at about 5.26 eV in case of mBJ. This study about the optoelectronic properties indicates that ZnAl 2 O 4 can be used in optical devices.
MODELING AND OPTIMIZATION OF THE CUTTING FLUID FLOW AND PARAMETERS FOR INCREASING TOOL LIFE IN SLOT MILLING ON St52
Authors: KHORASANI AMIR MAHYAR , KOOTSOOKOS ALEX
Published: November 2012

In this paper the CNC machining of St52 was modeled using an artificial neural network (ANN) in the form of a four-layer multi-layer perceptron (MLP). The cutting parameters used in the model were cutting fluid flow, feed rate, spindle speed and the depth of cut and the model output was the tool life. For obtaining more accuracy and spending less time Taguchi design of experiment (DOE) has been used and correlation between the output of the ANN and the experimental results was 96%. Further optimization process has been done by use of a genetic algorithm (GA). After optimization process tool life was increased about 8% equal to 33 min and was corroborated by experimental tests. This demonstrates that the coupling of an ANN with the GA optimization technique is a valid and useful approach to use.
Transient Population Inversion Induced and Probed by a Signal Pulse in a Continuous-Wave-Pumped Erbium-Doped Fiber Amplifier
Authors: Kuroda Keiji , Yoshikuni Yuzo
Published: November 2012

We report on the transient population inversion in a continuous-wave (CW)-pumped erbium-doped fiber amplifier. The temporal profile of the population inversion of the erbium-doped fiber amplifier is probed by a signal pulse that induces a decrease in the population inversion. The pump and signal power dependences of the profile are measured. We analyze the dependences by applying an analytical formula describing the temporal evolution of the population inversion at the input end of the fiber.
Pulse Excitation Method of Coherent-Population-Trapping Suitable for Chip-Scale Atomic Clock
Authors: Yano Yuichiro , Goka Shigeyoshi
Published: November 2012

We propose a pulse excitation method of coherent population trapping (CPT) with a liquid crystal optical modulator. Since liquid crystals enable reductions in size, weight, cost, and power consumption compared with acousto-optical modulators (AOMs), our method is suitable for chip-scale atomic clocks (CSACs). Experiments showed that pulse excitation with a liquid crystal modulator can narrow the CPT resonance linewidth and reduce the light shift effect using a 87Rb gas cell and the D1-line vertical-cavity surface-emitting laser (VCSEL). The CPT resonance linewidth and light shift sensitivity were less than one-eighth and one-third those for continuous excitation, respectively. They also showed that our method is comparable to that based on an AOM.
To Study the Effect of Paclitaxel on the Cytoplasmic Viscosity of Murine Macrophage Immune Cell RAW 264.7 Using Self-Developed Optical Tweezers System
Authors: Chen Ying-chun , Wu Chien-ming
Published: November 2012

In recent years, optical tweezers have become one of the tools to measure the mechanical properties of living cells. In this study, we first constructed an optical tweezers to investigate the cytoplasmic viscosity of immune cells. In addition to measuring viscosity of cells in a normal condition, we also treated cells with anti-cancer drug, Paclitaxel, and in order to study its effect on the cytoplasmic viscosity. The results showed that the viscosity decreased dramatically during the first 3 h. After 3 h, the change started to slow down and it remained nearly flat by the end of the experiment. In addition, we used the confocal laser scanning microscope to observe the cytoskeleton of the cell after drug treatment for 3 and 5 h, respectively, and found that actin filaments were disrupted and that the nucleus had disintegrated in some drug-treated cells, similar to the process of apoptosis. This study presents a new way for measuring the changes in cytoplasmic viscosity, and to determine if a cell is going into apoptosis as a result of a drug treatment.
Impact of site-specific phosphorylation of protein kinase A sites Ser23and Ser24of cardiac troponin I in human cardiomyocytes
Authors: Wijnker Paul J. M. , Foster D. Brian , Tsao Allison L. , Frazier Aisha H. , dos Remedios Cristobal G. , Murphy Anne M. , Stienen Ger J. M. , van der Velden Jolanda
Published: November 2012

PKA-mediated phosphorylation of contractile proteins upon β-adrenergic stimulation plays an important role in the regulation of cardiac performance. Phosphorylation of the PKA sites (Ser23/Ser24) of cardiac troponin (cTn)I results in a decrease in myofilament Ca2+sensitivity and an increase in the rate of relaxation. However, the relation between the level of phosphorylation of the sites and the functional effects in the human myocardium is unknown. Therefore, site-directed mutagenesis was used to study the effects of phosphorylation at Ser23and Ser24of cTnI on myofilament function in human cardiac tissue. Serines were replaced by aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. cTnI-DD mimics both sites phosphorylated, cTnI-AD mimics Ser23unphosphorylated and Ser24phosphorylated, cTnI-DA mimics Ser23phosphorylated and Ser24unphosphorylated, and cTnI-AA mimics both sites unphosphorylated. Force development was measured at various Ca2+concentrations in permeabilized cardiomyocytes in which the endogenous troponin complex was exchanged with these recombinant human troponin complexes. In donor cardiomyocytes, myofilament Ca2+sensitivity (pCa50) was significantly lower in cTnI-DD (pCa50: 5.39 ± 0.01) compared with cTnI-AA (pCa50: 5.50 ± 0.01), cTnI-AD (pCa50: 5.48 ± 0.01), and cTnI-DA (pCa50: 5.51 ± 0.01) at ∼70% cTn exchange. No effects were observed on the rate of tension redevelopment. In cardiomyocytes from idiopathic dilated cardiomyopathic tissue, a linear decline in pCa50with cTnI-DD content was observed, saturating at ∼55% bisphosphorylation. Our data suggest that in the human myocardium, phosphorylation of both PKA sites on cTnI is required to reduce myofilament Ca2+sensitivity, which is maximal at ∼55% bisphosphorylated cTnI. The implications for in vivo cardiac function in health and disease are detailed in the discussion in this article.
Deletion of 1–43 amino acids in cardiac myosin essential light chain blunts length dependency of Ca2+sensitivity and cross-bridge detachment kinetics
Authors: Michael John Jeshurun , Gollapudi Sampath K. , Ford Steven J. , Kazmierczak Katarzyna , Szczesna-Cordary Danuta , Chandra Murali
Published: November 2012

The role of cardiac myosin essential light chain (ELC) in the sarcomere length (SL) dependency of myofilament contractility is unknown. Therefore, mechanical and dynamic contractile properties were measured at SL 1.9 and 2.2 μm in cardiac muscle fibers from two groups of transgenic (Tg) mice: 1) Tg-wild-type (WT) mice that expressed WT human ventricular ELC and 2) Tg-Δ43 mice that expressed a mutant ELC lacking 1–43 amino acids. In agreement with previous studies, Ca2+-activated maximal tension decreased significantly in Tg-Δ43 fibers. pCa50(−log10[Ca2+]freerequired for half maximal activation) values at SL of 1.9 μm were 5.64 ± 0.02 and 5.70 ± 0.02 in Tg-WT and Tg-Δ43 fibers, respectively. pCa50values at SL of 2.2 μm were 5.70 ± 0.01 and 5.71 ± 0.01 in Tg-WT and Tg-Δ43 fibers, respectively. The SL-mediated increase in the pCa50value was statistically significant only in Tg-WT fibers ( P < 0.01), indicating that the SL dependency of myofilament Ca2+sensitivity was blunted in Tg-Δ43 fibers. The SL dependency of cross-bridge (XB) detachment kinetics was also blunted in Tg-Δ43 fibers because the decrease in XB detachment kinetics was significant ( P < 0.001) only at SL 1.9 μm. Thus the increased XB dwell time at the short SL augments Ca2+sensitivity at short SL and thus blunts SL-mediated increase in myofilament Ca2+sensitivity. Our data suggest that the NH2-terminal extension of cardiac ELC not only augments the amplitude of force generation, but it also may play a role in mediating the SL dependency of XB detachment kinetics and myofilament Ca2+sensitivity.
Identification of a region of rat chromosome 1 that impairs the myogenic response and autoregulation of cerebral blood flow in fawn-hooded hypertensive rats
Authors: Pabbidi Mallikarjuna R. , Juncos Julio , Juncos Luis , Renic Marija , Tullos Hurtis J. , Lazar Jozeph. , Jacob Howard J. , Harder David R. , Roman Richard J.
Published: November 2012

This study examined the effects of transfer of a 2.4-Mbp region of rat chromosome 1 (RNO1) from Brown Norway (BN) into fawn-hooded hypertensive (FHH) rats on autoregulation (AR) of cerebral blood flow (CBF) and the myogenic response of middle cerebral arteries (MCAs). AR of CBF was poor in FHH and FHH.1BNAR−congenic strains that excluded the critical 2.4-Mbp region. In contrast, AR was restored in FHH.1BNAR+congenic strains that included this region. The diameter of MCAs of FHH rats increased from 140 ± 14 to 157 ± 18 μm when transmural pressure was increased from 40 to 140 mmHg, but it decreased from 137 ± 5 to 94 ± 7 μm in FHH.1BNAR+congenic strains. Transient occlusion of MCAs reduced CBF by 80% in all strains. However, the hyperemic response following ischemia was significantly greater in FHH and AR−rats than that seen in AR+congenic strains (AR−, 173 ± 11% vs. AR+, 124 ± 5%). Infarct size and edema formation were also significantly greater in an AR−strain (38.6 ± 2.6 and 12.1 ± 2%) than in AR+congenic strains (27.6 ± 1.8 and 6.5 ± 0.9%). These results indicate that there is a gene in the 2.4-Mbp region of RNO1 that alters the development of myogenic tone in cerebral arteries. Transfer of this region from BN to FHH rats restores AR of CBF and vascular reactivity and reduces cerebral injury after transient occlusion and reperfusion of the MCA.
Human airway ciliary dynamics
Authors: Sears Patrick R. , Thompson Kristin , Knowles Michael R. , Davis C. William
Published: November 2012

Airway cilia depend on precise changes in shape to transport the mucus gel overlying mucosal surfaces. The ciliary motion can be recorded in several planes using video microscopy. However, cilia are densely packed, and automated computerized systems are not available to convert these ciliary shape changes into forms that are useful for testing theoretical models of ciliary function. We developed a system for converting planar ciliary motions recorded by video microscopy into an empirical quantitative model, which is easy to use in validating mathematical models, or in examining ciliary function, e.g., in primary ciliary dyskinesia (PCD). The system we developed allows the manipulation of a model cilium superimposed over a video of beating cilia. Data were analyzed to determine shear angles and velocity vectors of points along the cilium. Extracted waveforms were used to construct a composite waveform, which could be used as a standard. Variability was measured as the mean difference in position of points on individual waveforms and the standard. The shapes analyzed were the end-recovery, end-effective, and fastest moving effective and recovery with mean (± SE) differences of 0.31(0.04), 0.25(0.06), 0.50(0.12), 0.50(0.10), μm, respectively. In contrast, the same measures for three different PCD waveforms had values far outside this range.
Accelerated structural decrements in the aging female rhesus macaque lung compared with males
Authors: Herring Matt J. , Avdalovic Mark V. , Quesenberry Cheryl L. , Putney Lei F. , Tyler Nancy K. , Ventimiglia Frank F. , St. George Judith A. , Hyde Dallas M.
Published: November 2012

Aging is associated with morphometric changes in the lung that lead to decreased lung function. The nonhuman primate lung has been shown to have similar architectural, morphological, and developmental patterns to that of humans. We hypothesized that the lungs of rhesus monkeys age in a pattern similar to human lungs. Thirty-four rhesus monkeys from the California National Primate Research Center were euthanized, necropsied, and the whole lungs sampled. Stereological analysis was performed to assess the morphological changes associated with age. The number of alveoli declined significantly from age 9 to 33 yr with a greater decline in females compared with males. Lungs of females contained roughly 20% more alveoli at age 9 yr than males, but by ∼30 yr of age, females had 30% fewer alveoli than males. The volume of alveolar air also showed a significant linear decrease in females relative to age, while males did not. The number-weighted mean volume of alveoli showed a significant positive correlation with age in females but not in males. The volume of alveolar duct showed a significant positive correlation with age in females, but not in males. Structural decrements due to aging in the lung were increased in the female compared with male rhesus monkey.
Seasonal restructuring of the ground squirrel gut microbiota over the annual hibernation cycle
Authors: Carey Hannah V. , Walters William A. , Knight Rob
Published: November 2012

Many hibernating mammals suspend food intake during winter, relying solely on stored lipids to fuel metabolism. Winter fasting in these species eliminates a major source of degradable substrates to support growth of gut microbes, which may affect microbial community structure and host-microbial interactions. We explored the effect of the annual hibernation cycle on gut microbiotas using deep sequencing of 16S rRNA genes from ground squirrel cecal contents. Squirrel microbiotas were dominated by members of the phyla Bacteroidetes, Firmicutes, and Verrucomicrobia. UniFrac analysis showed that microbiotas clustered strongly by season, and maternal influences, diet history, host age, and host body temperature had minimal effects. Phylogenetic diversity and numbers of operational taxonomic units were lowest in late winter and highest in the spring after a 2-wk period of refeeding. Hibernation increased relative abundance of Bacteroidetes and Verrucomicrobia, phyla that contain species capable of surviving on host-derived substrates such as mucins, and reduced relative abundance of Firmicutes, many of which prefer dietary polysaccharides. Hibernation reduced cecal short-chain fatty acid and ammonia concentrations, and increased and decreased concentrations of acetate and butyrate, respectively. These results indicate that the ground squirrel microbiota is restructured each year in a manner that reflects differences in microbial preferences for dietary vs. host-derived substrates, and thus the competitive abilities of different taxa to survive in the altered environment in the hibernator gut.
Isolation of interstitial fluid and demonstration of local proinflammatory cytokine production and increased absorptive gradient in chronic peritoneal dialysis
Authors: Rosengren Bert-Inge , Sagstad Solfrid J. , Karlsen Tine V. , Wiig Helge
Published: November 2012

In peritoneal dialysis (PD) patients, the frequent exposure to “unphysiological” dialysis fluids elicits a chronic state of a low-grade peritoneal inflammation leading to interstitial matrix remodeling and angiogenesis. Proinflammatory cytokines are important regulators involved in this inflammatory process that ultimately leads to dysfunction of the peritoneum as a dialysis membrane. We aimed to measure the local concentrations of proinflammatory cytokines in the peritoneal interstitial fluid (IF). Furthermore, we wanted to assess how the driving forces for fluid and solute exchanges are affected in a remodeled interstitial matrix and thus measured the colloid osmotic pressure (COP) gradient in rats that were exposed to chronic PD. After 8 wk of peritoneal dialysis, IF from peritoneum was isolated using a centrifugation method, and was analyzed for cytokine content and COP along with plasma. For several of the proinflammatory cytokines there were gradients from IF to plasma, showing local production. For some cytokines, the concentration in IF was increased severalfold, whereas IL-18 was increased systemically due to PD. Furthermore, the presence of the catheter per se seemed to increase cytokine levels. COP in IF was significantly decreased in the PD group, while collagen and hyaluronan content was increased. Collectively, our data suggest that the increased levels of proinflammatory cytokines after PD may be an integral component of the development of fibrosis and angiogenesis commonly seen in PD patients, and the decreased COP in IF after chronic PD may shift the Starling equilibrium across peritoneal capillaries to an absorptive state.
Alterations in upper limb muscle synergy structure in chronic stroke survivors
Authors: Roh Jinsook , Rymer William Z. , Perreault Eric J. , Yoo Seng Bum , Beer Randall F.
Published: November 2012

Previous studies in neurologically intact subjects have shown that motor coordination can be described by task-dependent combinations of a few muscle synergies, defined here as a fixed pattern of activation across a set of muscles. Arm function in severely impaired stroke survivors is characterized by stereotypical postural and movement patterns involving the shoulder and elbow. Accordingly, we hypothesized that muscle synergy composition is altered in severely impaired stroke survivors. Using an isometric force matching protocol, we examined the spatial activation patterns of elbow and shoulder muscles in the affected arm of 10 stroke survivors (Fugl-Meyer <25/66) and in both arms of six age-matched controls. Underlying muscle synergies were identified using non-negative matrix factorization. In both groups, muscle activation patterns could be reconstructed by combinations of a few muscle synergies (typically 4). We did not find abnormal coupling of shoulder and elbow muscles within individual muscle synergies. In stroke survivors, as in controls, two of the synergies were comprised of isolated activation of the elbow flexors and extensors. However, muscle synergies involving proximal muscles exhibited consistent alterations following stroke. Unlike controls, the anterior deltoid was coactivated with medial and posterior deltoids within the shoulder abductor/extensor synergy and the shoulder adductor/flexor synergy in stroke was dominated by activation of pectoralis major, with limited anterior deltoid activation. Recruitment of the altered shoulder muscle synergies was strongly associated with abnormal task performance. Overall, our results suggest that an impaired control of the individual deltoid heads may contribute to poststroke deficits in arm function.