bluuebunny/crossref_metadata_2025 huggingface dataset:
Grain‐Boundary Sliding in a Superplastic Three‐Phase Alumina–Zirconia–Mullite Ceramic Composite
Authors: Hoo Christopher M. , Men Danju , Taherabadi Lili , Mecartney Martha L.
Published: March 2011

Relative grain‐boundary sliding at the surface of a three‐phase superplastic ceramic after compressive deformation was measured using atomic force microscopy and scanning electron microscopy to investigate the x, y, and z strain contributions at different types of grain boundaries. This alumina (40 vol%), zirconia (30 vol%), and mullite (30 vol%) composite had an average grain size of 260 nm. The composite was deformed in compression under constant stress conditions in sequential increments of 5%–10% true strain at 1350°C. Six different types of grain boundaries (alumina/alumina, zirconia/zirconia, mullite/mullite, alumina/zirconia, alumina/mullite, and zirconia/mullite) were characterized, for over 800 boundaries in all. It was found that all types of boundaries contributed significantly to the overall deformation with no differentiation between grain boundaries of the same phase and grain boundaries of dissimilar phases, with the exception of mullite/mullite boundaries that had a more complex surface topology due to the in situ formation of mullite during sintering.
The effect of agriculture on repayment efficiency: a look at MFI borrowing groups
Authors: Raghunathan Uthra K. , Escalante Cesar L. , Dorfman Jeffrey H. , Ames Glenn C. W. , Houston Jack E.
Published: March 2011

There is a widely held industry assumption from microfinance institutions that agricultural loans have poorer repayment rates, which has resulted in many loans being provided for small businesses as opposed to agricultural purposes. Using data from a sample of 100 borrowing groups from a south Indian Microfinance Institutions (MFI), this study challenges this belief by analyzing the repayment efficiency of borrowing groups and reflects on the implications for agricultural microfinance loans. The analysis is run using Bayesian stochastic frontier estimation with an exponential hierarchical prior on the efficiency term. Our results indicate that the average efficiency of the borrowing groups analyzed was approximately 75% and that having a higher percentage of agricultural loans increased borrowing group efficiency while gains in efficiency also rose as the size of the borrowing group increases.
Migration of bone marrow‐derived cells and improved perfusion after treatment with erythropoietin in a murine model of myocardial infarction
Authors: Brunner Stefan , Huber Bruno C. , Weinberger Tobias , Vallaster Marcus , Wollenweber Tim , Gerbitz Armin , Hacker Marcus , Franz Wolfgang‐Michael
Published: March 2011

Erythropoietin (EPO) was shown to have protective effects after myocardial infarction (MI) by neovascularization and antiapoptotic mechanisms. Beside direct receptor‐dependent mechanisms, mobilization and homing of bone marrow‐derived cells (BMCs) may play a pivotal role in this regard. In this study, we intended to track different subpopulations of BMCs and to assess serially myocardial perfusion changes in EPO‐treated mice after MI. To allow tracking of BMCs, we used a chimeric mouse model. Therefore, mice (C57BL/6J) were sublethally irradiated, and bone marrow (BM) from green fluorescent protein transgenic mice was transplanted. Ten weeks later coronary artery ligation was performed to induce MI. EPO was injected for 3 days with a total dose of 5000 IU/kg. Subpopulations (CD31, c‐kit, CXCR‐4 and Sca‐1) of EGFP+ cells were studied in peripheral blood, bone marrow and hearts by flow cytometry. Myocardial perfusion was serially investigated in vivo by pinhole single‐photon emission computed tomography (SPECT) at days 6 and 30 after MI. EPO‐treated animals revealed an enhanced mobilization of BMCs into peripheral blood. The numbers of these cells in BM remained unchanged. Homing of all BMCs subpopulations to the ischaemic myocardium was significantly increased in EPO‐treated mice. Among the investigated subpopulations, EPO predominantly affected migration of CXCR‐4+ (4.3‐fold increase). Repetitively SPECT analyses revealed a reduction of perfusion defects after EPO treatment over time. Our study shows that EPO treatment after MI enhances the migration capacity of BMCs into ischaemic tissue, which may attribute to an improved perfusion and reduced size of infarction, respectively.
α‐Lipoic acid attenuates vascular calcification via reversal of mitochondrial function and restoration of Gas6/Axl/Akt survival pathway
Authors: Kim Hyunsoo , Kim Han‐Jong , Lee Kyunghee , Kim Jin‐Man , Kim Hee Sun , Kim Jae‐Ryong , Ha Chae‐Myeong , Choi Young‐Keun , Lee Sun Joo , Kim Joon‐Young , Harris Robert A. , Jeong Daewon , Lee In‐Kyu
Published: March 2011

Vascular calcification is prevalent in patients with chronic kidney disease and leads to increased cardiovascular morbidity and mortality. Although several reports have implicated mitochondrial dysfunction in cardiovascular disease and chronic kidney disease, little is known about the potential role of mitochondrial dysfunction in the process of vascular calcification. This study investigated the effect of α‐lipoic acid (ALA), a naturally occurring antioxidant that improves mitochondrial function, on vascular calcification in vitro and in vivo. Calcifying vascular smooth muscle cells (VSMCs) treated with inorganic phosphate (Pi) exhibited mitochondrial dysfunction, as demonstrated by decreased mitochondrial membrane potential and ATP production, the disruption of mitochondrial structural integrity and concurrently increased production of reactive oxygen species. These Pi‐induced functional and structural mitochondrial defects were accompanied by mitochondria‐dependent apoptotic events, including release of cytochrome c from the mitochondria into the cytosol, subsequent activation of caspase‐9 and ‐3, and chromosomal DNA fragmentation. Intriguingly, ALA blocked the Pi‐induced VSMC apoptosis and calcification by recovery of mitochondrial function and intracellular redox status. Moreover, ALA inhibited Pi‐induced down‐regulation of cell survival signals through the binding of growth arrest‐specific gene 6 (Gas6) to its cognate receptor Axl and subsequent Akt activation, resulting in increased survival and decreased apoptosis. Finally, ALA significantly ameliorated vitamin D3‐induced aortic calcification and mitochondrial damage in mice. Collectively, the findings suggest ALA attenuates vascular calcification by inhibiting VSMC apoptosis through two distinct mechanisms; preservation of mitochondrial function via its antioxidant potential and restoration of the Gas6/Axl/Akt survival pathway.
Inhibition of keratin 17 expression with antisense and RNAi strategies: exploring novel therapy for psoriasis
Authors: Chang Ting , Sun Linchao , Wang Yan , Wang Datai , Li Wei , Li Chunying , Gao Tianwen , Liu Yufeng , Wang Gang
Published: March 2011

Abstract:  Psoriasis is now considered to be a chronic, immune‐mediated and inflammatory skin disease. As the precise cause of psoriasis remains unknown, its treatment is challenging for dermatologists. Keratin 17 (K17), an intermediate filament protein, is highly expressed in psoriatic lesions, while not normally expressed in healthy epidermis. Studies have suggested that K17 plays a role in the pathogenesis of psoriasis. However, no study has been performed to determine the potential application of K17 down‐regulation as a treatment option for psoriatic lesions. We hypothesized that anti‐K17 interference may suppress the development and progression of psoriasis and potentially serve as a novel strategy for the treatment of psoriasis. Therefore, we down‐regulated and silenced K17 gene expression in keratinocytes (KCs) using antisense and RNA interference (RNAi) techniques. We found that K17‐specific antisense oligonucleotides (ASODN) or siRNAs inhibited proliferation and induced apoptosis in KCs as well as down‐regulated K17 expression at both mRNA and protein levels. For our in vivo study, we constructed the SCID‐hu xenogeneic transplantation psoriasis mouse model by grafting psoriatic lesions onto SCID mice and topically applied K17‐specific ASODN and liposome‐encapsulated siRNA to the grafts. We observed morphological and histological improvement in the treated psoriatic grafts. As a result, K17 mRNA and protein expression was significantly decreased in the grafts of the mouse model. Taken together, we conclude that anti‐K17 therapy is an effective treatment option for psoriasis, and the K17 molecule, as a new target, may hold tremendous potential for the treatment of psoriasis in the future.
Climate‐driven population dynamics of a North American freshwater fish, Ictalurus furcatus: inference from the mitochondrial control region sequence data
Authors: Padhi Abinash
Published: March 2011

Abstract –  Using the Bayesian coalescent methodology and mitochondrial control region sequence data, the present study reconstructs the dynamics of effective population size of a temperature‐driven seasonal migratory North American freshwater fish, Ictalurus furcatus, during the last glacial cycle. The trend in effective population size is directly associated with the trend in surface air temperature; it is inversely associated with the accumulation of ice sheets in North America during the contemporary period. The long‐term decrease in effective population size during the cold period followed by rebound during the warmer period could be explained by this species’ preference for relatively warmer temperatures for its optimal growth and survival.
Turbidity‐induced changes in emergent effects of multiple predators with different foraging strategies
Authors: VanLandeghem Matthew M. , Carey Michael P. , Wahl David H.
Published: March 2011

Abstract –  In natural systems, prey frequently interact with multiple predators and the outcome often cannot be predicted by summing the effects of individual predator species. Multiple predator interactions can create emergent effects for prey, but how those change across environmental gradients is poorly understood. Turbidity is an environmental factor in aquatic systems that may influence multiple predator effects on prey. Interactions between a cruising predator (largemouth bass Micropterus salmoides) and an ambush predator (muskellunge Esox masquinongy) and their combination foraging on a shared prey (bluegill Lepomis macrochirus) were examined across a turbidity gradient. Turbidity modified multiple predator effects on prey. In clear water, combined predators consumed in total more prey than expected from individual predator treatments, suggesting risk enhancement for prey. In moderately turbid water, the predators consumed fewer prey together than expected, suggesting a risk reduction for prey. At high turbidity, there were no apparent emergent effects; however, the cruising predator consumed more prey than the ambush predator, suggesting an advantage for this predator. Understanding multiple predator traits across a gradient of turbidity increases our understanding of how complex natural systems function.
Allometric patterns of fetal head growth in mysticetes and odontocetes: Comparison of Balaena mysticetus and Stenella attenuata
Authors: Armfield Brooke A. , George J. Craig , Vinyard Christopher J. , Thewissen J. G. M.
Published: March 2011

Abstract

Unlike other mammals, odontocetes and mysticetes have highly derived craniofacial bones. A growth process referred to as “telescoping” is partly responsible for this morphology. Here, we explore how changes in facial morphology during fetal growth relate to differences in telescoping between the adult odontocete Stenella attenuata and the mysticete Balaena mysticetus. We conclude that in both Stenella and Balaena head size increases allometrically. Similarly, odontocete nasal length and mysticete mouth size have strong positive allometry compared to total body length. However, the differences between odontocetes and mysticetes in telescoping are not directly associated with their fetal growth patterns. Our results suggest that cranial changes related to echolocation and feeding between odontocetes and mysticetes, respectively, begin during ontogeny before telescoping is initiated.
Reduction in the environmental impact of waste discharged by fish farms through feed and feeding
Authors: Amirkolaie Abdolsamad K.
Published: March 2011

The discharge of waste from aquaculture operations can lead to eutrophication and destruction of natural ecosystem in receiving water body. A controlled waste production strategy is necessary to maintain sustainable aquaculture growth into the future. As feed is the major source of waste in aquaculture, the management of aquaculture waste should be approached through diet formulation or feeding strategies. Highly digestible diets have been introduced as a solution to reduce solid waste excretion. Further reductions in solid waste can be achieved through careful selection of feed ingredients and feed processing to improve nutrient availability. An increase in faeces consistency by diet manipulation can improve solid removal efficiency. This condition can reduce the proportion of solids in discharged water in the effluent and also improve farm water quality. A reduction in dissolved nitrogen waste can be achieved by ensuring a balance between protein and energy causing fish to use non‐protein sources as energy. Phosphorous waste can be decreased through careful ingredient selection and proper processing to improve digestibility. A proper feed ration and feeding method for each species should be adopted because feed waste constitutes a large part of waste production.
Development and characterisation of 20 microsatellite loci isolated from the large bent‐wing bat, Miniopterus schreibersii (Chiroptera: Miniopteridae) and their cross‐taxa utility in the family Miniopteridae
Authors: WOOD REBECCA , WEYENETH NICOLE , APPLETON BELINDA
Published: March 2011

The large bent‐wing bat, Miniopterus schreibersii (Kuhl 1819), has a long history of taxonomic uncertainty and many populations are known to be in a state of decline. Microsatellite loci were developed for the taxonomic and population genetic assessment of the Australian complex of this species. Of the 33 primer sets designed for this research, seven (21%) were deemed suitably polymorphic for population‐level analyses of the Australian taxa, with five (71%) of these loci revealing moderate to high levels of polymorphism (PIC = 0.56 to 0.91). The cross‐taxa utility of the M. schreibersii microsatellite markers was assessed in the microbat (Chiroptera) family Miniopteridae. Sub‐species and species covering the Miniopteridae’s global distribution (with the exception of the Middle East) were selected, numbering 25 taxa in total. Amplification was successful for 26 loci, of which 20 (77%) were polymorphic. High cross‐taxa utility of markers was observed with amplification achieved for all taxa for between four (20%) and 20 (100%) loci, and polymorphism was considered moderate to high (PIC = 0.47–0.91) for 12 (60%) of these loci. The high cross‐taxa utility of the microsatellites reported herein reveal versatile and cost‐effective molecular markers, contributing an important genetic resource for the research and conservation of Miniopteridae species worldwide.
Regulation of NR4A nuclear receptor expression by oncogenic BRAF in melanoma cells
Authors: Smith Aaron G. , Lim Wen , Pearen Michael , Muscat George E. O. , Sturm Richard A.
Published: March 2011

Activating mutations in the MAPK pathway effectors, NRAS or BRAF, are detected in over 70% of melanomas. Accordingly, the identification of downstream targets of constitutive MAPK signalling in melanoma represents a major goal in understanding the genesis of this disease. We report here the regulation of members of the NR4A family of nuclear receptors by the BRAF‐MEK‐ERK cascade in melanoma cells. Expression profiling of melanoma cells in which both the NR4A1 and NR4A2 family members have been down‐regulated by siRNA revealed alterations in genes associated with proliferation, survival and invasiveness of tumour cells. Notably, the up‐regulation of Wnt/β‐catenin pathway antagonists, DACT1 and CITED1, following NR4A1/2 ablation suggests a possible link between NR4A and β‐catenin activity in melanoma cells. Taken together, these data suggest that dysregulation of NR4A nuclear receptors expression and function by the MAPK pathway may contribute to melanoma tumourigenicity.
Enhanced dissolution and stability of adefovir dipivoxil by cocrystal formation
Authors: Gao Yuan , Zu Hui , Zhang Jianjun
Published: March 2011

Objectives

The objectives of this study were to prepare and characterize the novel adefovir dipivoxil–saccharin cocrystal and to demonstrate the enhanced dissolution and stability of adefovir dipivoxil by cocrystal formation.

Methods

Adefovir dipivoxil–saccharin cocrystal was prepared using a novel solution crystallization approach and scaled up to 30 g for subsequent studies. DSC, IR and XRPD were used to characterize the novel solid form. The stoichiometry of the cocrystal was analysed by HPLC. Dissolution and chemical stability were assessed and compared with marketed adefovir dipivoxil (form 1) used in marketed Hepsera Tablets.

Key findings

A new solid adefovir dipivoxil–saccharin cocrystal with unique melting point, DSC, FTIR and XRPD data was obtained. The molar ratio of adefovir dipivoxil and saccharin in the cocrystal was determined to be 1 : 1. The cocrystal had a pH-independent dissolution profile and showed a two-fold increase in the dissolution efficiency in water and phosphate buffer (pH 6.8) compared with adefovir dipivoxil. The cocrystal was kinetically much more stable than form 1. Form 1 degraded almost completely at 60°C in 18 days, while adefovir dipivoxil–saccharin cocrystal remained unchanged for 47 days at 60°C.

Conclusions

This study demonstrated that the dissolution and stability of adefovir dipivoxil could be significantly enhanced by its cocrystal formation with saccharin. The use of cocrystals could be a feasible and valuable approach for improving the physicochemical properties of adefovir dipivoxil.
Assessment of biological activity of novel peptide analogues of angiotensin IV
Authors: Gard Paul R , Olivier George , Golding Bruno , Bourner Claire , Dang Tony , Haliru Haliru , Higgins Eleanor , Kimberley Helen , Loginova Olga , Madhavi Shahnaz , Ryan Denis
Published: March 2011

Objectives

Angiotensin IV (Ang IV) is a metabolite of angiotensin II which acts on specific AT4 receptors identified as the enzyme insulin regulated aminopeptidase (IRAP). The transduction process of these receptors is unresolved, but Ang IV inhibits the aminopeptidase activity. Ang IV improves cognition in animal models thus there is a desire to develop metabolically stable analogues for further development.

Methods

Peptide analogues of Ang IV were obtained commercially or synthesised. Each peptide was tested in vitro for its ability to inhibit the aminopeptidase activity (IRAP) of mouse brain homogenates and for its effects on isolated rat uterine smooth muscle.

Key findings

[Des-Val1]-Ang IV, acetylated-Ang IV-amide, Ang IV-amide and [des-His4]-Ang IV all inhibited IRAP. [Sar1, Ile8]-Angiotensin II (10 µm) had an effect greater than that of Ang IV or any of the other analogues studied. In isolated uterine smooth muscle, angiotensins II and IV induced contractions, which could be antagonised by an AT1-receptor antagonist. None of the novel peptides induced uterine smooth muscle contractions, but [Sar1, des Arg2-Gly8]-angiotensin II showed significant antagonism of the contractile effects of angiotensin II and carboxyamide-terminated Ang IV-NH2 showed antagonism of Ang IV-induced contractions.

Conclusions

This study provides five novel inhibitors of IRAP worthy of assessment in behavioural models of learning and memory. The analogues are devoid of AT1 receptor agonist properties, and the carboxyamide analogue presents an opportunity to elucidate the mechanism of action of Ang IV as, like Ang IV, it inhibits IRAP, but antagonises the effects of Ang IV on isolated smooth muscle.
Entrapment of 5-fluorouracil into PLGA matrices using supercritical antisolvent processes
Authors: Kalantarian Pardis , Haririan Ismaeil , Najafabadi Abdolhossein Rouholamini , Shokrgozar Mohammad Ali , Vatanara Alireza
Published: March 2011

Objectives

Two different supercritical antisolvent processes were performed to co-precipitate 5-fluorouracil (5-FU) and poly(lactide-co-glycolide) simultaneously. 5-FU is a hydrophilic antitumor agent, and is more effective when administered at a lower dose for a longer period of time.

Methods

Controlled-release polymeric systems of 5-FU were produced, and morphology, thermal behavior, in-vitro release and cytotoxicity of microparticles were analysed.

Key findings

Dissolution studies showed that 33% of drug was released in 21 days, which represents a long-lasting profile. To evaluate the efficacy of the released drug on cancer cells, the MTT assay cytotoxicity test was performed using human lung carcinoma A549 cell lines. There was no significant difference between the cell inhibition rates of the released drug and unprocessed 5-FU at the same drug concentration level. IC50 values were 69.12 mg/ml for unprocessed 5-FU and 68.71 mg/ml for the released drug.

Conclusions

Application of supercritical processing for co-precipitation of 5-FU and PLGA provided mild and non-aqueous conditions, so the hydrophilic drug incorporated in the polymer had good stability during the process.
Excitatory–inhibitory imbalance in hypoglossal neurons during the critical period of postnatal development in the rat
Authors: Gao Xiu‐ping , Liu Qing‐song , Liu Qiuli , Wong‐Riley Margaret T. T.
Published: March 2011

Non‐technical summary  Previously, our laboratory has reported that within a narrow window toward the end of the second postnatal week in a rat's life, many sudden neurochemical and functional changes occurred that rendered the animal less responsive to decreased oxygen supply. The present study extends these findings to the recordings of single nerve cells and documents that, during this critical period of normal postnatal respiratory network development, the responses to excitation are significantly reduced and those to inhibition are significantly enhanced. Thus, the system is under greater inhibitory influence at this time. These findings may have significant implication for understanding sudden infant death syndrome, whose victims are seemingly normal infants with no known pathologies. The peak incidence of SIDS is not at birth, but between the second and fourth months after birth, a theoretical critical period of postnatal respiratory development.
Action potential bursts enhance transmitter release at a giant central synapse
Authors: Zhang Bo , Sun Liang , Yang Yi‐Mei , Huang Hong‐Ping , Zhu Fei‐Peng , Wang Li , Zhang Xiao‐Yu , Guo Shu , Zuo Pan‐Li , Zhang Claire X. , Ding Jiu‐Ping , Wang Lu‐Yang , Zhou Zhuan
Published: March 2011

Non‐technical summary  Information is coded in the form of bursts of electrical impulses propagating among nerve cells which form complex networks in the brain. Effective communication between these cells depends on the ability for cross‐talk among them through release and reception of chemical substances (neurotransmitters). This study uses the hearing system as a model to show that the patterns of electrical impulses can dramatically impact the amount of neurotransmitter released. When presented in short clusters, these impulses are more effective in releasing neurotransmitters than those composed of the same number of impulses but given continuously. Our findings may potentially help us understand how nerve cells code and transfer information in the mammalian brain, and in particular, how auditory neurons localize the sound source in space.
Changes in muscle sympathetic nerve activity and vascular responses evoked in the spinotrapezius muscle of the rat by systemic hypoxia
Authors: Hudson Steven , Johnson Christopher D. , Marshall Janice M.
Published: March 2011

Non‐technical summary  Hitherto, activity in sympathetic vasoconstrictor nerves that supply arterial vessels of skeletal muscle has been deduced from recordings made on mixed nerves that may be supplying skin, muscle or other tissue. We describe new methodology that allows direct recordings from nerve fibres on the surface of arterial vessels of muscle. The impulses occurred irregularly, but with rhythms that reflected heart rate and breathing rate. When oxygen levels in the blood were progressively lowered (hypoxia), muscle sympathetic nerve activity progressively increased from an average impulse frequency of 0.2 to one of 0.62 per second. Simultaneously, heart rate and respiratory rate increased, while our recordings of blood flow into the muscle showed that the arterial vessels dilated. We deduce that hypoxia causes an increase in the activity of the sympathetic nerves that supply muscle blood vessels that is mainly dependent on the stimulus for breathing, but their normal vasoconstrictor effect is overcome by local dilator influences.
Olfactory receptor neuron responses coding for rapid odour sampling
Authors: Ghatpande Ambarish S. , Reisert Johannes
Published: March 2011

Non‐technical summary  Odorants are transported into the nasal cavity upon air inhalation where they are detected by olfactory receptor neurons (ORNs), which transduce the odorant molecules into action potentials. The rate of stimulation thus depends on the chosen breathing frequency, which in mice ranges from 2 to 10 Hz. This poses the question how ORNs respond to rapidly changing stimulation rates. Individual mouse ORNs respond reliably to repetitive 2 Hz stimulations resembling normal breathing, but actually perform much poorer when the stimulation rate is increased to 5 Hz, which is more akin to sniffing. In this case, rarely more than 50% of the stimulations elicit any response, with an increase in odorant concentration further reducing the response rate, becoming zero at high concentrations. This counterintuitive observation can be understood in the framework of an adaptive filter, which allows the animal to selectively alter its ORN output depending on the chosen breathing rate.
Cell type‐specific and activity‐dependent dynamics of action potential‐evoked Ca2+ signals in dendrites of hippocampal inhibitory interneurons
Authors: Evstratova Alesya , Chamberland Simon , Topolnik Lisa
Published: March 2011

Non‐technical summary  Action potentials generated at the level of the cell body can propagate back to neuronal dendrites, where they activate different types of voltage‐sensitive calcium channels and produce massive calcium influx. Although these calcium signals may control dendritic integration, their mechanisms, dynamic properties and role in different cell types remain largely unknown. We found that in dendrites of hippocampal interneurons, an inhibitory cell type involved in control of network excitability, specific types of calcium channels are present but are recruited in an activity‐dependent manner. Furthermore, their activation produces calcium rises spatially restricted to proximal dendritic sites, where they control the efficacy of transmission at inhibitory synapses. The pathway by which this happens appears to constitute a negative feedback loop – increased firing activity of interneurons potentiates the inhibitory drive that they receive, thus decreasing the activity of interneurons further. This may have a profound effect on the recruitment of interneurons and network activity.
Population Pharmacokinetic and Pharmacodynamic Analysis of Linezolid and a Hematologic Side Effect, Thrombocytopenia, in Japanese Patients
Authors: Sasaki Tomohiro , Takane Hiroshi , Ogawa Katsuhiro , Isagawa Sayaka , Hirota Takeshi , Higuchi Shun , Horii Toshinobu , Otsubo Kenji , Ieiri Ichiro
Published: March 2011

Linezolid is an antimicrobial agent to treat infections by Gram-positive pathogens, including methicillin-resistant
Staphylococcus aureus
(MRSA). While effective, linezolid treatment frequently is associated with hematological side effects, especially thrombocytopenia. However, little is known about the mechanism of this side effect and the exposure-response relationship. The present population pharmacokinetic/pharmacodynamic (PPK/PD) study was undertaken to elucidate the factors that determine linezolid levels, the relationship between exposure to linezolid and a decrease in platelet counts, and appropriate dosage adjustments based on exposure levels. In total, 50 patients (135 plasma samples) were used for the PPK analysis. The PPK analysis revealed that renal function and severe liver cirrhosis (Child Pugh grade C) significantly affect the pharmacokinetics of linezolid according to the equation clearance (liter/h) = 2.85 × (creatinine clearance/60.9)
0.618
× 0.472
CIR
(CIR indicates cirrhosis status; 0 for noncirrhosis, 1 for cirrhosis patients). Using 603 platelet counts from 45 patients, a PPK/PD analysis with a semimechanistic pharmacodynamic model described the relationship between linezolid exposure and platelet counts quantitatively, and the newly constructed model was validated using external data (776 platelet counts from 60 patients). Simulation indicated considerable risks in patients with insufficient renal function (creatinine clearance, ≤30 ml/min) or severe liver cirrhosis. For these patients, a reduced dosage (600 mg/day) would be recommended for sufficient efficacy (area under the concentration-time curve over 24 h in the steady state divided by the MIC, >100) and safety.
Identification of Pyruvate Kinase in Methicillin-Resistant Staphylococcus aureus as a Novel Antimicrobial Drug Target
Authors: Zoraghi Roya , See Raymond H. , Axerio-Cilies Peter , Kumar Nag S. , Gong Huansheng , Moreau Anne , Hsing Michael , Kaur Sukhbir , Swayze Richard D. , Worrall Liam , Amandoron Emily , Lian Tian , Jackson Linda , Jiang Jihong , Thorson Lisa , Labriere Christophe , Foster Leonard , Brunham Robert C. , McMaster William R. , Finlay B. Brett , Strynadka Natalie C. , Cherkasov Artem , Young Robert N. , Reiner Neil E.
Published: March 2011

Novel classes of antimicrobials are needed to address the challenge of multidrug-resistant bacteria such as methicillin-resistantStaphylococcus aureus(MRSA). Using the architecture of the MRSA interactome, we identified pyruvate kinase (PK) as a potential novel drug target based upon it being a highly connected, essential hub in the MRSA interactome. Structural modeling, including X-ray crystallography, revealed discrete features of PK in MRSA, which appeared suitable for the selective targeting of the bacterial enzyme.In silicolibrary screening combined with functional enzymatic assays identified an acyl hydrazone-based compound (IS-130) as a potent MRSA PK inhibitor (50% inhibitory concentration [IC50] of 0.1 μM) with >1,000-fold selectivity over human PK isoforms. Medicinal chemistry around the IS-130 scaffold identified analogs that more potently and selectively inhibited MRSA PK enzymatic activity andS. aureusgrowthin vitro(MIC of 1 to 5 μg/ml). These novel anti-PK compounds were found to possess antistaphylococcal activity, including both MRSA and multidrug-resistantS. aureus(MDRSA) strains. These compounds also exhibited exceptional antibacterial activities against other Gram-positive genera, including enterococci and streptococci. PK lead compounds were found to be noncompetitive inhibitors and were bactericidal. In addition, mutants with significant increases in MICs were not isolated after 25 bacterial passages in culture, indicating that resistance may be slow to emerge. These findings validate the principles of network science as a powerful approach to identify novel antibacterial drug targets. They also provide a proof of principle, based upon PK in MRSA, for a research platform aimed at discovering and optimizing selective inhibitors of novel bacterial targets where human orthologs exist, as leads for anti-infective drug development.
INX-08189, a Phosphoramidate Prodrug of 6-
O
-Methyl-2′-
C
-Methyl Guanosine, Is a Potent Inhibitor of Hepatitis C Virus Replication with Excellent Pharmacokinetic and Pharmacodynamic Properties
Authors: Vernachio John H. , Bleiman Blair , Bryant K. Dawn , Chamberlain Stanley , Hunley Damound , Hutchins Jeff , Ames Brenda , Gorovits Elena , Ganguly Babita , Hall Andrea , Kolykhalov Alexander , Liu Yule , Muhammad Jerry , Raja Nicholas , Walters C. Robin , Wang Jin , Williams Karen , Patti Joseph M. , Henson Geoffrey , Madela Karolina , Aljarah Mohamed , Gilles Arnaud , McGuigan Christopher
Published: March 2011

INX-08189 is an aryl-phosphoramidate of 6-
O
-methyl-2′-
C
-methyl guanosine. INX-08189 was highly potent in replicon assays, with a 50% effective concentration of 10 ± 6 nM against hepatitis C genotype 1b at 72 h. The inhibitory effect on viral replication was rapid, with a 50% effective concentration (EC
50
) of 35 ± 8 nM at 24 h. An intracellular 2′-
C
-methyl guanosine triphosphate (2′-
C
-MeGTP) concentration of 2.43 ± 0.42 pmol/10
6
cells was sufficient to achieve 90% inhibition of viral replication.
In vitro
resistance studies confirmed that the S282T mutation in the NS5b gene conferred an approximately 10-fold reduction in sensitivity to INX-08189. However, the complete inhibition of S282T mutant replicons still could be achieved with an EC
90
of 344 ± 170 nM. Drug combination studies of INX-08189 and ribavirin indicated significant synergy in antiviral potency both in wild-type and S282T-expressing replicons. Genotype 1b replicons could be cleared after 14 days of culture when exposed to as little as 20 nM INX-08189. No evidence of mitochondrial toxicity was observed after 14 days of INX-08189 exposure in both HepG2 and CEM human cell lines.
In vivo
studies of rats and cynomolgus monkeys demonstrated that 2′-
C
-MeGTP concentrations in liver equivalent to the EC
90
could be attained after a single oral dose of INX-08189. Rat liver 2′-
C
-MeGTP concentrations were proportional to dose, sustained for greater than 24 h, and correlated with plasma concentrations of the nucleoside metabolite 2′-
C
-methyl guanosine. The characteristics displayed by INX-08189 support its continued development as a clinical candidate for the treatment of chronic HCV infection.
Ligand-Dependent Disorder of the Ω Loop Observed in Extended-Spectrum SHV-Type β-Lactamase
Authors: Sampson Jared M. , Ke Wei , Bethel Christopher R. , Pagadala S. R. R. , Nottingham Michael D. , Bonomo Robert A. , Buynak John D. , van den Akker Focco
Published: March 2011

Among Gram-negative bacteria, resistance to β-lactams is mediated primarily by β-lactamases (EC 3.2.6.5), periplasmic enzymes that inactivate β-lactam antibiotics. Substitutions at critical amino acid positions in the class A β-lactamase families result in enzymes that can hydrolyze extended-spectrum cephalosporins, thus demonstrating an “extended-spectrum” β-lactamase (ESBL) phenotype. Using SHV ESBLs with substitutions in the Ω loop (R164H and R164S) as target enzymes to understand this enhanced biochemical capability and to serve as a basis for novel β-lactamase inhibitor development, we determined the spectra of activity and crystal structures of these variants. We also studied the inactivation of the R164H and R164S mutants with tazobactam and SA2-13, a unique β-lactamase inhibitor that undergoes a distinctive reaction chemistry in the active site. We noted that the reduced

K
i

values for the R164H and R164S mutants with SA2-13 are comparable to those with tazobactam (submicromolar). The apo enzyme crystal structures of the R164H and R164S SHV variants revealed an ordered Ω loop architecture that became disordered when SA2-13 was bound. Important structural alterations that result from the binding of SA2-13 explain the enhanced susceptibility of these ESBL enzymes to this inhibitor and highlight ligand-dependent Ω loop flexibility as a mechanism for accommodating and hydrolyzing β-lactam substrates.
Evaluation of CLSI M44-A2 Disk Diffusion and Associated Breakpoint Testing of Caspofungin and Micafungin Using a Well-Characterized Panel of Wild-Type and
fks
Hot Spot Mutant Candida Isolates
Authors: Arendrup Maiken Cavling , Park Steven , Brown Steven , Pfaller Michael , Perlin David S.
Published: March 2011

Disk diffusion testing has recently been standardized by the CLSI, and susceptibility breakpoints have been established for several antifungal compounds. For caspofungin, 5-μg disks are approved, and for micafungin, 10-μg disks are under evaluation. We evaluated the performances of caspofungin and micafungin disk testing using a panel of
Candida
isolates with and without known
FKS
echinocandin resistance mechanisms. Disk diffusion and microdilution assays were performed strictly according to CLSI documents M44-A2 and M27-A3. Eighty-nine clinical
Candida
isolates were included:
Candida albicans
(20 isolates/10 mutants),
C. glabrata
(19 isolates/10 mutants),
C. dubliniensis
(2 isolates/1 mutant),
C. krusei
(16 isolates/3 mutants),
C. parapsilosis
(14 isolates/0 mutants), and
C. tropicalis
(18 isolates/4 mutants). Quality control strains were
C. parapsilosis
ATCC 22019 and
C. krusei
ATCC 6258. The correlations between zone diameters and MIC results were good for both compounds, with identical susceptibility classifications for 93.3% of the isolates by applying the current CLSI breakpoints. However, the numbers of
fks
hot spot mutant isolates misclassified as being susceptible (S) (very major errors [VMEs]) were high (61% for caspofungin [S, ≥11 mm] and 93% for micafungin [S, ≥14 mm]). Changing the disk diffusion breakpoint to S at ≥22 mm significantly improved the discrimination. For caspofungin, 1 VME was detected (a
C. tropicalis
isolate with an F76S substitution) (3.5%), and for micafungin, 10 VMEs were detected, the majority of which were for
C. glabrata
(8/10). The broadest separation between zone diameter ranges for wild-type (WT) and mutant isolates was seen for caspofungin (6 to 12 mm versus −4 to 7 mm). In conclusion, caspofungin disk diffusion testing with a modified breakpoint led to excellent separation between WT and mutant isolates for all
Candida
species.
Selection of Clinically Relevant Protease Inhibitor-Resistant Viruses Using the Genotype 2a Hepatitis C Virus Infection System
Authors: Cheng Guofeng , Chan Katie , Yang Huiling , Corsa Amy , Pokrovskii Maria , Paulson Matthew , Bahador Gina , Zhong Weidong , Delaney William
Published: March 2011

Treatment of patients infected with hepatitis C virus (HCV) with direct acting antivirals can lead to the emergence of drug-resistant variants that may pose a long-term threat to viral eradication. HCV replicons have been used to select resistance mutations; however, genotype 2a JFH-1-based viruses provide the opportunity to perform resistance selection in a
bona fide
infection system. In this study, we used a tissue culture-adapted J6/JFH-1 virus to select resistance to the NS3 protease inhibitors BILN-2061 and VX-950. Lunet-CD81 cells were infected with J6/JFH-1 virus and maintained in the presence of inhibitors until high-titer viral supernatant was produced. Viral supernatants were passaged over naive cells at escalating drug concentrations, and the resulting viruses were then characterized. Three NS3 resistance mutations were identified in BILN-2061-resistant viruses: A156G, D168A, and D168V. Interestingly, D168A, D168V, and A156T/V, but not A156G, were selected in parallel using a genotype 2a replicon. For VX-950, the T54A and A156S NS3 resistance mutations were identified in the virus selections, whereas only A156T/V emerged in genotype 2a replicon selections. Of note, VX-950 resistance mutations selected using the 2a virus (T54A and A156S) were also observed during VX-950 clinical studies in genotype 2 patients. We also performed viral fitness evaluations and determined that the mutations selected in the viral system did not confer marked reductions in virus production kinetics or peak titers. Overall, the HCV infection system is an efficient tool for drug resistance selections and has advantages for the rapid identification and characterization of clinically relevant resistance mutations.
Overexpression of AmpC and Efflux Pumps in Pseudomonas aeruginosa Isolates from Bloodstream Infections: Prevalence and Impact on Resistance in a Spanish Multicenter Study
Authors: Cabot Gabriel , Ocampo-Sosa Alain A. , Tubau Fe , Macia María D. , Rodríguez Cristina , Moya Bartolomé , Zamorano Laura , Suárez Cristina , Peña Carmen , Martínez-Martínez Luis , Oliver Antonio ,   the Spanish Network for Research in Infectious Diseases (REIPI)
Published: March 2011

The prevalence and impact of the overexpression of AmpC and efflux pumps were evaluated with a collection of 190
Pseudomonas aeruginosa
isolates recovered from bloodstream infections in a 2008 multicenter study (10 hospitals) in Spain. The MICs of a panel of 13 antipseudomonal agents were determined by microdilution, and the expressions of
ampC
,
mexB
,
mexY
,
mexD
, and
mexF
were determined by real-time reverse transcription (RT)-PCR. Up to 39% of the isolates overexpressed at least one of the mechanisms.
ampC
overexpression (24.2%) was the most prevalent mechanism, followed by
mexY
(13.2%),
mexB
(12.6%),
mexF
(4.2%), and
mexD
(2.2%). The overexpression of
mexB
plus
mexY
, documented for 5.3% of the isolates, was the only combination showing a significantly (
P
= 0.02) higher prevalence than expected from the frequencies of the individual mechanisms (1.6%). Additionally, all imipenem-resistant isolates studied (25 representative isolates) showed inactivating mutations in
oprD
. Most of the isolates nonsusceptible to piperacillin-tazobactam (96%) and ceftazidime (84%) overexpressed
ampC
, while
mexB
(25%) and
mexY
(29%) overexpressions gained relevance among cefepime-nonsusceptible isolates. Nevertheless, the prevalence of
mexY
overexpression was highest among tobramycin-nonsusceptible isolates (37%), and that of
mexB
was highest among meropenem-nonsusceptible isolates (33%). Regarding ciprofloxacin-resistant isolates, besides the expected increased prevalence of efflux pump overexpression, a highly significant link to
ampC
overexpression was documented for the first time: up to 52% of ciprofloxacin-nonsusceptible isolates overexpressed
ampC
, sharply contrasting with the 24% documented for the complete collection (
P
< 0.001). In summary, mutation-driven resistance was frequent in
P. aeruginosa
isolates from bloodstream infections, whereas metallo-β-lactamases, detected in 2 isolates (1%) producing VIM-2, although with increasing prevalences, were still uncommon.
In VitroandIn VivoAntiplasmodial Activities of Risedronate and Its Interference with Protein Prenylation in Plasmodium falciparum
Authors: Jordão Fabiana Morandi , Saito Alexandre Yukio , Miguel Danilo Ciccone , de Jesus Peres Valnice , Kimura Emília Akemi , Katzin Alejandro Miguel
Published: March 2011

The increasing resistance of malarial parasites to almost all available drugs calls for the identification of new compounds and the detection of novel targets. Here, we establish the antimalarial activities of risedronate, one of the most potent bisphosphonates clinically used to treat bone resorption diseases, against blood stages ofPlasmodium falciparum(50% inhibitory concentration [IC50] of 20.3 ± 1.0 μM). We also suggest a mechanism of action for risedronate against the intraerythrocytic stage ofP. falciparumand show that protein prenylation seems to be modulated directly by this drug. Risedronate inhibits the transfer of the farnesyl pyrophosphate group to parasite proteins, an effect not observed for the transfer of geranylgeranyl pyrophosphate. Ourin vivoexperiments further demonstrate that risedronate leads to an 88.9% inhibition of the rodent parasitePlasmodium bergheiin mice on the seventh day of treatment; however, risedronate treatment did not result in a general increase of survival rates.
Analysis of the Borrelia burgdorferi Cyclic-di-GMP-Binding Protein PlzA Reveals a Role in Motility and Virulence
Authors: Pitzer Joshua E. , Sultan Syed Z. , Hayakawa Yoshihiro , Hobbs Gerry , Miller Michael R. , Motaleb Md A.
Published: March 2011

The cyclic-dimeric-GMP (c-di-GMP)-binding protein PilZ has been implicated in bacterial motility and pathogenesis. Although BB0733 (PlzA), the only PilZ domain-containing protein in
Borrelia burgdorferi
, was reported to bind c-di-GMP, neither its role in motility or virulence nor it's affinity for c-di-GMP has been reported. We determined that PlzA specifically binds c-di-GMP with high affinity (dissociation constant [

K
d

], 1.25 μM), consistent with

K
d

values reported for c-di-GMP-binding proteins from other bacteria. Inactivation of the monocistronically transcribed
plzA
resulted in an opaque/solid colony morphology, whereas the wild-type colonies were translucent. While the swimming pattern of mutant cells appeared normal, on swarm plates, mutant cells exhibited a significantly reduced swarm diameter, demonstrating a role of
plzA
in motility. Furthermore, the
plzA
mutant cells were significantly less infectious in experimental mice (as determined by 50% infectious dose [ID
50
]) relative to wild-type spirochetes. The mutant also had survival rates in fed ticks lower than those of the wild type. Consequently,
plzA
mutant cells failed to complete the mouse-tick-mouse infection cycle, indicating
plzA
is essential for the enzootic life cycle of
B. burgdorferi
. All of these defects were corrected when the mutant was complemented in
cis
. We propose that failure of
plzA
mutant cells to infect mice was due to altered motility; however, the possibility that an unidentified factor(s) contributed to interruption of the
B. burgdorferi
enzootic life cycle cannot yet be excluded.
Regulation of Trypanosoma cruzi-Induced Myocarditis by Programmed Death Cell Receptor 1
Authors: Gutierrez Fredy R. S. , Mariano Flávia S. , Oliveira Carlo J. F. , Pavanelli Wander R. , Guedes Paulo M. M. , Silva Grace K. , Campanelli Ana P. , Milanezi Cristiane M. , Azuma Miyuki , Honjo Tasuku , Teixeira Mauro M. , Aliberti Julio C. S. , Silva João S.
Published: March 2011

Trypanosoma cruziinfection causes intense myocarditis, leading to cardiomyopathy and severe cardiac dysfunction. Protective adaptive immunity depends on balanced signaling through a T cell receptor and coreceptors expressed on the T cell surface. Such coreceptors can trigger stimulatory or inhibitory signals after binding to their ligands in antigen-presenting cells (APC).T. cruzimodulates the expression of coreceptors in lymphocytes after infection. Deregulated inflammation may be due to unbalanced expression of these molecules. Programmed death cell receptor 1 (PD-1) is a negative T cell coreceptor that has been associated with T cell anergy or exhaustion and persistent intracellular infections. We aimed to study the role of PD-1 duringT. cruzi-induced acute myocarditis in mice. Cytometry assays showed that PD-1 and its ligands are strongly upregulated in lymphocytes and APC in response toT. cruziinfectionin vivoandin vitro. Lymphocytes infiltrating the myocardium exhibited high levels of expression of these molecules. An increased cardiac inflammatory response was found in mice treated with blocking antibodies against PD-1, PD-L1, and to a lesser extent, PD-L2, compared to that found in mice treated with rat IgG. Similar results in PD-1−/−mice were obtained. Moreover, the PD-1 blockade/deficiency led to reduced parasitemia and tissue parasitism but increased mortality. These results suggest the participation of a PD-1 signaling pathway in the control of acute myocarditis induced byT. cruziand provide additional insight into the regulatory mechanisms in the pathogenesis of Chagas' disease.