bluuebunny/crossref_metadata_2025 huggingface dataset:
Temporal response of positive and negative regulators in response to acute and chronic exercise training in mice
Authors: Olenich Sara A. , Gutierrez‐Reed Navarre , Audet Gerald N. , Olfert I. Mark
Published: July 2013

Key points

Angiogenic regulators respond to acute exercise with different temporal expression patterns (e.g. 2–4 h versus 12–24 h) creating a complex multifaceted response that must be considered in studies using a single time point for post‐exercise analyses.

In response to chronic training there appears to be a complex coordination in the proteomic responses of both positive and negative angiogenic factors that correspond with training‐induced muscle capillary adaptation, such that altered basal expression and acute responses to exercise appear to withdraw or reduce the stimulus of angiogenic regulators in an expanding capillary bed with active angiogenesis.

These are the first data to show that nucleolin (a protein responsible for transcriptional processing and transportation of proteins from the cytoplasm to the nucleus) is responsive to acute exercise. We speculate that nucleolin may work in concert with vascular endothelial growth factor‐A (VEGF) and endostatin.

Temporal responses observed in mice, particularly for VEGF, MMP‐2 and MMP‐9, may not be directly comparable to humans.

Abstract  Angiogenesis is controlled by a balance between positive and negative angiogenic factors, but temporal protein expression of many key angiogenic regulators in response to exercise are still poorly defined. In C57BL/6 mice, we evaluated the temporal protein expression of several pro‐angiogenic and anti‐angiogenic factors in response to (1) a single acute bout of exercise and (2) chronic exercise training resulting from 3, 5, 7, 14 and 28 days of voluntary wheel running. Following acute exercise, protein levels of vascular endothelial growth factor‐A (VEGF), endostatin and nucleolin were increased at 2–4 h (P < 0.05), whereas matrix metalloproteinase (MMP)‐2 was elevated within a 12–24 h window (P < 0.05). Training increased muscle capillarity 11%, 15% and 22% starting with 7, 14 and 28 days of training, respectively (P < 0.01). Basal VEGF and MMP‐2 were increased by 31% and 22%, respectively, compared to controls (P < 0.05) after 7 days (7d) training, but decreased to back to baseline after 14d training. After 28d training VEGF fell 49% below baseline control (P < 0.01). Basal muscle expression of thrombospondin 1 (TSP‐1) was ∼900% greater in 14d‐ and 28d‐trained mice compared to either 5d‐ and 7d‐trained mice (P < 0.05), and tended to increase by ∼180–258% compared to basal control levels (P < 0.10). The acute responsiveness of VEGF to exercise in untrained mice (i.e. 161% increase, P < 0.001) was lost with capillary adaptation occurring after 7, 14 and 28d training. Taken together, these data support the notion that skeletal muscle angiogenesis is controlled by a balance between positive and negative mitogens, and reveals a complex, highly‐coordinated, temporal scheme whereby these factors can differentially influence capillary growth in response to acute versus chronic exercise.
A membrane glucocorticoid receptor mediates the rapid/non‐genomic actions of glucocorticoids in mammalian skeletal muscle fibres
Authors: Pérez María Hernández‐Alcalá , Cormack Jonathan , Mallinson David , Mutungi Gabriel
Published: July 2013

Key points

Glucocorticoids are stress hormones used in the treatment of many chronic inflammatory diseases including asthma. They exert most of their physiological/pharmacological actions by regulating the activity of genes involved in the inflammatory response. However, they also have rapid/non‐genomic effects whose functions are poorly understood.

In this study we used two widely prescribed glucocorticoids, beclomethasone dipropionate and prednisolone acetate, to investigate whether these hormones have rapid/non‐genomic effects in mammalian skeletal muscles.

Both glucocorticoids increased maximum force in slow‐twitch muscle fibres/cells without significantly affecting that of fast‐twitch muscle fibres.

The increase in force occurred within 10 min and was blocked by an inhibitor of the glucocorticoid receptor and a protein (antibody) that binds the receptor.

These findings suggest that these hormones/drugs have rapid/non‐genomic effects in mammalian skeletal muscles; these effects are mediated by a membrane glucocorticoid receptor and are physiologically/pharmacologically beneficial, especially in slow muscles.

Abstract  Glucocorticoids (GCs) are steroid hormones released from the adrenal gland in response to stress. They are also some of the most potent anti‐inflammatory and immunosuppressive drugs currently in clinical use. They exert most of their physiological and pharmacological actions through the classical/genomic pathway. However, they also have rapid/non‐genomic actions whose physiological and pharmacological functions are still poorly understood. Therefore, the primary aim of this study was to investigate the rapid/non‐genomic effects of two widely prescribed glucocorticoids, beclomethasone dipropionate (BDP) and prednisolone acetate (PDNA), on force production in isolated, intact, mouse skeletal muscle fibre bundles. The results show that the effects of both GCs on maximum isometric force (Po) were fibre‐type dependent. Thus, they increased Po in the slow‐twitch fibre bundles without significantly affecting that of the fast‐twitch fibre bundles. The increase in Po occurred within 10 min and was insensitive to the transcriptional inhibitor actinomycin D. Also, it was maximal at ∼250 nm and was blocked by the glucocorticoid receptor (GCR) inhibitor RU486 and a monoclonal anti‐GCR, suggesting that it was mediated by a membrane (m) GCR. Both muscle fibre types expressed a cytosolic GCR. However, a mGCR was present only in the slow‐twitch fibres. The receptor was more abundant in oxidative than in glycolytic fibres and was confined mainly to the periphery of the fibres where it co‐localised with laminin. From these findings we conclude that the rapid/non‐genomic actions of GCs are mediated by a mGCR and that they are physiologically/therapeutically beneficial, especially in slow‐twitch muscle fibres.
The working stroke of the myosin II motor in muscle is not tightly coupled to release of orthophosphate from its active site
Authors: Caremani Marco , Melli Luca , Dolfi Mario , Lombardi Vincenzo , Linari Marco
Published: July 2013

Key points

Force and shortening in muscle are caused by ATP‐driven working strokes of myosin II motors, during their cyclic interactions with the actin filament in each half‐sarcomere. Crystallographic studies indicate that the working stroke consists in an interdomain movement of the myosin motor associated with the release of inorganic phosphate (Pi).

Here the coupling of the working stroke with the release of Pi is studied in situ using fast half‐sarcomere mechanics on skinned fibres from rabbit psoas.

The isotonic velocity transient following stepwise force reductions superimposed on isometric contraction measures the mechanical manifestation of the working stroke and its rate of regeneration.

The results indicate that the release of Pi from the catalytic site of an actin‐attached myosin motor can occur at any stage of the working stroke, and a myosin motor uses two consecutive actin monomers to maximize the power during shortening.

Abstract  Skeletal muscle shortens faster against a lower load. This force–velocity relationship is the fundamental determinant of muscle performance in vivo and is due to ATP‐driven working strokes of myosin II motors, during their cyclic interactions with the actin filament in each half‐sarcomere. Crystallographic studies suggest that the working stroke is associated with the release of phosphate (Pi) and consists of 70 deg tilting of a light‐chain domain that connects the catalytic domain of the myosin motor to the myosin tail and filament. However, the coupling of the working stroke with Pi release is still an unsolved question. Using nanometre–microsecond mechanics on skinned muscle fibres, we impose stepwise drops in force on an otherwise isometric contraction and record the isotonic velocity transient, to measure the mechanical manifestation of the working stroke of myosin motors and the rate of its regeneration in relation to the half‐sarcomere load and [Pi]. We show that the rate constant of the working stroke is unaffected by [Pi], while the subsequent transition to steady velocity shortening is accelerated. We propose a new chemo‐mechanical model that reproduces the transient and steady state responses by assuming that: (i) the release of Pi from the catalytic site of a myosin motor can occur at any stage of the working stroke, and (ii) a myosin motor, in an intermediate state of the working stroke, can slip to the next actin monomer during filament sliding. This model explains the efficient action of muscle molecular motors working as an ensemble in the half‐sarcomere.
Regulation of membrane trafficking by signalling on endosomal and lysosomal membranes
Authors: Li Xinran , Garrity Abigail G. , Xu Haoxing
Published: July 2013

Abstract  Endosomal and lysosomal membrane trafficking requires the coordination of multiple signalling events to control cargo sorting and processing, and endosome maturation. The initiation and termination of signalling events in endosomes and lysosomes is not well understood, but several key regulators have been identified, which include small GTPases, phosphoinositides, and Ca2+. Small GTPases act as master regulators and molecular switches in a GTP‐dependent manner, initiating signalling cascades to regulate the direction and specificity of endosomal trafficking. Phosphoinositides are membrane‐bound lipids that indicate vesicular identities for recruiting specific cytoplasmic proteins to endosomal membranes, thus allowing specificity of membrane fusion, fission, and cargo sorting to occur within and between specific vesicle compartments. In addition, phosphoinositides regulate the function of membrane proteins such as ion channels and transporters in a compartment‐specific manner to mediate transport and signalling. Finally, Ca2+, a locally acting second messenger released from intracellular ion channels, may provide precise spatiotemporal regulation of endosomal signalling and trafficking events. Small GTPase signalling can regulate phosphoinositide conversion during endosome maturation, and electrophysiological studies on isolated endosomes have shown that endosomal and lysosomal Ca2+ channels are directly modulated by endosomal lipids. Thus trafficking and maturation of endosomes and lysosomes can be precisely regulated by dynamic changes in GTPases and membrane lipids, as well as Ca2+ signalling. Importantly, impaired phosphoinositide and Ca2+ signalling can cause endosomal and lysosomal trafficking defects at the cellular level, and a spectrum of lysosome storage diseases.
Intrauterine inflammation alters fetal cardiopulmonary and cerebral haemodynamics in sheep
Authors: Galinsky Robert , Hooper Stuart B. , Polglase Graeme R. , Moss Timothy J. M.
Published: July 2013

Key points

Intrauterine inflammation impairs fetal pulmonary vascular development and increases cerebral metabolism in fetal sheep. Whether these structural and metabolic changes have functional consequences for fetal cardiopulmonary and cerebral haemodynamics is presently unknown.

We demonstrated that intra‐amniotic administration of lipopolysaccharide increased fetal pulmonary vascular resistance, and reduced pulmonary blood flow and carotid arterial oxygen content in the fetus and caused a transient increase in fetal cerebral blood flow. These effects occurred over a time course consistent with previously observed effects on pulmonary vascular development and cerebral metabolism.

Our data suggest that pathophysiological changes in cardiopulmonary and cerebral haemodynamics observed in fetuses exposed to intrauterine inflammation may be present and detectable in human pregnancies, offering potential for detecting fetuses affected by intrauterine inflammation.

Abstract  Intrauterine inflammation impairs fetal pulmonary vascular development and increases cerebral metabolic rate in fetal sheep. We hypothesized that these structural and metabolic effects of intrauterine inflammation would be accompanied by reduced fetal pulmonary blood flow and increased cerebral perfusion. Fetal sheep were instrumented at 112 days of gestation (term is 147 days) for measurement of cardiopulmonary and cerebral haemodynamics. At 118 days ewes were randomly assigned to receive intra‐amniotic lipopolysaccharide (LPS, 20 mg from Escherichia coli; n= 7) or saline (control, 4 ml; n= 6). Fetal haemodynamic data were recorded continually from 1 h before intra‐amniotic LPS or saline, until 144 h after. Fetal arterial blood was sampled before, and periodically after, intra‐amniotic LPS or saline. End‐diastolic and mean pulmonary blood flows were significantly lower than control from 48 and 96 h after LPS exposure, respectively, until the end of the experiment. Carotid blood flow was transiently increased at 96 and 120 h after LPS exposure. Carotid arterial oxygen content was lower than control from 48 h after intra‐amniotic LPS. Fetal arterial lactate concentration was higher than control between 4 and 12 h after intra‐amniotic LPS. Experimental intrauterine inflammation reduces pulmonary blood flow in fetal sheep, over a time course consistent with impaired pulmonary vascular development. Increased carotid blood flow after LPS administration may reflect an inflammation‐induced increase in cerebral metabolic demand.
Contraction‐induced lipolysis is not impaired by inhibition of hormone‐sensitive lipase in skeletal muscle
Authors: Alsted Thomas J. , Ploug Thorkil , Prats Clara , Serup Annette K. , Høeg Louise , Schjerling Peter , Holm Cecilia , Zimmermann Robert , Fledelius Christian , Galbo Henrik , Kiens Bente
Published: July 2013

Key points

In skeletal muscle hormone‐sensitive lipase (HSL) is considered the only enzyme responsible for breakdown of intramyocellular triacylglycerol (IMTG) during contractions. This notion is based on indirect measures in which important cellular events are not taken into account.

Using two histochemical techniques to measure breakdown of IMTG during contractions in isolated skeletal muscles we found that IMTG was decreased (1) in rat muscles during acute pharmacological blockade of HSL, and (2) in muscles of HSL knockout mice.

We demonstrated that adipose triglyceride lipase (ATGL) and HSL collectively account for at least 98% of the total TG lipase activity in mouse muscle, and other TG lipases accordingly seem of negligible importance for breakdown of IMTG.

In conclusion, breakdown of IMTG occurs in the contracting muscle in the absence of HSL activity. Our data suggest that ATGL is activated during contractions and plays a major role in breakdown of IMTG.

Abstract  In skeletal muscle hormone‐sensitive lipase (HSL) has long been accepted to be the principal enzyme responsible for lipolysis of intramyocellular triacylglycerol (IMTG) during contractions. However, this notion is based on in vitro lipase activity data, which may not reflect the in vivo lipolytic activity. We investigated lipolysis of IMTG in soleus muscles electrically stimulated to contract ex vivo during acute pharmacological inhibition of HSL in rat muscles and in muscles from HSL knockout (HSL‐KO) mice. Measurements of IMTG are complicated by the presence of adipocytes located between the muscle fibres. To circumvent the problem with this contamination we analysed intramyocellular lipid droplet content histochemically. At maximal inhibition of HSL in rat muscles, contraction‐induced breakdown of IMTG was identical to that seen in control muscles (P < 0.001). In response to contractions IMTG staining decreased significantly in both HSL‐KO and WT muscles (P < 0.05). In vitro TG hydrolase activity data revealed that adipose triglyceride lipase (ATGL) and HSL collectively account for ∼98% of the TG hydrolase activity in mouse skeletal muscle, other TG lipases accordingly being of negligible importance for lipolysis of IMTG. The present study is the first to demonstrate that contraction‐induced lipolysis of IMTG occurs in the absence of HSL activity in rat and mouse skeletal muscle. Furthermore, the results suggest that ATGL is activated and plays a major role in lipolysis of IMTG during muscle contractions.
Genetic Characterization and Emergence of the Metallo-β-Lactamase GIM-1 in Pseudomonas spp. and Enterobacteriaceae during a Long-Term Outbreak
Authors: Wendel Andreas F. , Brodner Alexander H. B. , Wydra Stephan , Ressina Sofija , Henrich Birgit , Pfeffer Klaus , Toleman Mark A. , MacKenzie Colin R.
Published: July 2013

Since the first isolation in 2002, the metallo-β-lactamase GIM-1 has not been detected outside Germany. The data presented here, for 50 clinical
bla
GIM-1
-positive isolates, including
Pseudomonas
spp. and
Enterobacteriaceae
(
Enterobacter cloacae
,
Klebsiella oxytoca
,
Serratia marcescens
,
Escherichia coli
, and
Citrobacter freundii
), collected between 2007 and 2012 at the original site in an ongoing outbreak, demonstrate a diverse genetic background and dissemination of the gene conferring resistance to enteric bacteria.
Adjunctive
N
-Acetyl-
l
-Cysteine in Treatment of Murine Pneumococcal Meningitis
Authors: Högen Tobias , Demel Cornelia , Giese Armin , Angele Barbara , Pfister Hans-Walter , Koedel Uwe , Klein Matthias
Published: July 2013

Despite antibiotic therapy, acute and long-term complications are still frequent in pneumococcal meningitis. One important trigger of these complications is oxidative stress, and adjunctive antioxidant treatment with
N
-acetyl-
l
-cysteine was suggested to be protective in experimental pneumococcal meningitis. However, studies of effects on neurological long-term sequelae are limited. Here, we investigated the impact of adjunctive
N
-acetyl-
l
-cysteine on long-term neurological deficits in a mouse model of meningitis. C57BL/6 mice were intracisternally infected with
Streptococcus pneumoniae
. Eighteen hours after infection, mice were treated with a combination of ceftriaxone and placebo or ceftriaxone and
N
-acetyl-
l
-cysteine, respectively. Two weeks after infection, neurologic deficits were assessed using a clinical score, an open field test (explorative activity), a t-maze test (memory function), and auditory brain stem responses (hearing loss). Furthermore, cochlear histomorphological correlates of hearing loss were assessed. Adjunctive
N
-acetyl-
l
-cysteine reduced hearing loss after pneumococcal meningitis, but the effect was minor. There was no significant benefit of adjunctive
N
-acetyl-
l
-cysteine treatment in regard to other long-term complications of pneumococcal meningitis. Cochlear morphological correlates of meningitis-associated hearing loss were not reduced by adjunctive
N
-acetyl-
l
-cysteine. In conclusion, adjunctive therapy with
N
-acetyl-
l
-cysteine at a dosage of 300 mg/kg of body weight intraperitoneally for 4 days reduced hearing loss but not other neurologic deficits after pneumococcal meningitis in mice. These results make a clinical therapeutic benefit of
N
-acetyl-
l
-cysteine in the treatment of patients with pneumococcal meningitis questionable.
Interaction of Silver Nanoparticles with Serum Proteins Affects Their Antimicrobial Activity
In Vivo
Authors: Gnanadhas Divya Prakash , Ben Thomas Midhun , Thomas Rony , Raichur Ashok M. , Chakravortty Dipshikha
Published: July 2013

The emergence of multidrug-resistant bacteria is a global threat for human society. There exist recorded data that silver was used as an antimicrobial agent by the ancient Greeks and Romans during the 8th century. Silver nanoparticles (AgNPs) are of potential interest because of their effective antibacterial and antiviral activities, with minimal cytotoxic effects on the cells. However, very few reports have shown the usage of AgNPs for antibacterial therapy
in vivo
. In this study, we deciphered the importance of the chosen methods for synthesis and capping of AgNPs for their improved activity
in vivo
. The interaction of AgNPs with serum albumin has a significant effect on their antibacterial activity. It was observed that uncapped AgNPs exhibited no antibacterial activity in the presence of serum proteins, due to the interaction with bovine serum albumin (BSA), which was confirmed by UV-Vis spectroscopy. However, capped AgNPs [with citrate or poly(vinylpyrrolidone)] exhibited antibacterial properties due to minimized interactions with serum proteins. The damage in the bacterial membrane was assessed by flow cytometry, which also showed that only capped AgNPs exhibited antibacterial properties, even in the presence of BSA. In order to understand the
in vivo
relevance of the antibacterial activities of different AgNPs, a murine salmonellosis model was used. It was conclusively proved that AgNPs capped with citrate or PVP exhibited significant antibacterial activities
in vivo
against
Salmonella
infection compared to uncapped AgNPs. These results clearly demonstrate the importance of capping agents and the synthesis method for AgNPs in their use as antimicrobial agents for therapeutic purposes.
Improvement of the Pharmacokinetics and
In Vivo
Antibacterial Efficacy of a Novel Type IIa Topoisomerase Inhibitor by Formulation in Liposomes
Authors: Shapiro Adam B. , Newman Joseph , Goteti Kosalaram , Beaudoin Marie-Eve , Harrison Rane , Hopkins Sussie , Agrawal Nikunj , Rivin Olga
Published: July 2013

Several useful properties of liposome-based formulations of various existing antibacterial drugs have been reported. These properties include lower MICs, improved pharmacokinetics, lower toxicity, selective distribution to infected tissues, and enhanced
in vivo
efficacy. Here we report
in vivo
studies of a liposomal formulation of a member of a novel class of antibacterial type II topoisomerase inhibitors, others of which have progressed to early phases of clinical trials. The free (i.e., nonliposomal) compound has broad-spectrum MICs but suboptimal pharmacokinetics in rats and mice, characterized by a high volume of distribution and rapid clearance. The liposomal formulation of the compound had essentially unchanged MICs but greatly reduced volume of distribution and clearance in rats and mice. In an
in vivo
mouse model of
Staphylococcus aureus
infection of one thigh, the liposomal compound localized preferentially to the infected thigh, whereas the free compound showed no preference for the infected versus the uninfected thigh. Most importantly, the liposomal compound had enhanced efficacy at clearing the infection compared with the free compound. Delivery of this class of compounds as liposomal formulations may offer clinical advantages compared with free compounds.
Genomic Characterization of Ciprofloxacin Resistance in a Laboratory-Derived Mutant and a Clinical Isolate of Streptococcus pneumoniae
Authors: Lupien Andréanne , Billal Dewan S. , Fani Fereshteh , Soualhine Hafid , Zhanel George G. , Leprohon Philippe , Ouellette Marc
Published: July 2013

The broad-spectrum fluoroquinolone ciprofloxacin is a bactericidal antibiotic targeting DNA topoisomerase IV and DNA gyrase encoded by the
parC
and
gyrA
genes. Resistance to ciprofloxacin in
Streptococcus pneumoniae
mainly occurs through the acquisition of mutations in the quinolone resistance-determining region (QRDR) of the ParC and GyrA targets. A role in low-level ciprofloxacin resistance has also been attributed to efflux systems. To look into ciprofloxacin resistance at a genome-wide scale and to discover additional mutations implicated in resistance, we performed whole-genome sequencing of an
S. pneumoniae
isolate selected for resistance to ciprofloxacin
in vitro
(128 μg/ml) and of a clinical isolate displaying low-level ciprofloxacin resistance (2 μg/ml). Gene disruption and DNA transformation experiments with PCR fragments harboring the mutations identified in the
in vitro
S. pneumoniae
mutant revealed that resistance is mainly due to QRDR mutations in
parC
and
gyrA
and to the overexpression of the ABC transporters PatA and PatB. In contrast, no QRDR mutations were identified in the genome of the
S. pneumoniae
clinical isolate with low-level resistance to ciprofloxacin. Assays performed in the presence of the efflux pump inhibitor reserpine suggested that resistance is likely mediated by efflux. Interestingly, the genome sequence of this clinical isolate also revealed mutations in the coding region of
patA
and
patB
that we implicated in resistance. Finally, a mutation in the NAD(P)H-dependent glycerol-3-phosphate dehydrogenase identified in the
S. pneumoniae
clinical strain was shown to protect against ciprofloxacin-mediated reactive oxygen species.
Candida tropicalis Antifungal Cross-Resistance Is Related to Different Azole Target (Erg11p) Modifications
Authors: Forastiero A. , Mesa-Arango A. C. , Alastruey-Izquierdo A. , Alcazar-Fuoli L. , Bernal-Martinez L. , Pelaez T. , Lopez J. F. , Grimalt J. O. , Gomez-Lopez A. , Cuesta I. , Zaragoza O. , Mellado E.
Published: July 2013

Candida tropicalis
ranks between third and fourth among
Candida
species most commonly isolated from clinical specimens. Invasive candidiasis and candidemia are treated with amphotericin B or echinocandins as first-line therapy, with extended-spectrum triazoles as acceptable alternatives.
Candida tropicalis
is usually susceptible to all antifungal agents, although several azole drug-resistant clinical isolates are being reported. However,
C. tropicalis
resistant to amphotericin B is uncommon, and only a few strains have reliably demonstrated a high level of resistance to this agent. The resistance mechanisms operating in
C. tropicalis
strains isolated from clinical samples showing resistance to azole drugs alone or with amphotericin B cross-resistance were elucidated. Antifungal drug resistance was related to mutations of the azole target (Erg11p) with or without alterations of the ergosterol biosynthesis pathway. The antifungal drug resistance shown
in vitro
correlated very well with the results obtained
in vivo
using the model host
Galleria mellonella
. Using this panel of strains, the
G. mellonella
model system was validated as a simple, nonmammalian minihost model that can be used to study
in vitro-in vivo
correlation of antifungals in
C. tropicalis
. The development in
C. tropicalis
of antifungal drug resistance with different mechanisms during antifungal treatment has potential clinical impact and deserves specific prospective studies.
Pharmacokinetics and Absorption of Paromomycin and Gentamicin from Topical Creams Used To Treat Cutaneous Leishmaniasis
Authors: Ravis William R. , Llanos-Cuentas Alejandro , Sosa Nestor , Kreishman-Deitrick Mara , Kopydlowski Karen M. , Nielsen Carl , Smith Kirsten S. , Smith Philip L. , Ransom Janet H. , Lin Yuh-Jing , Grogl Max
Published: July 2013

This study evaluated the pharmacokinetics of topical creams containing 15% paromomycin (“paromomycin alone”) and 15% paromomycin plus 0.5% gentamicin (WR 279,396) in patients with cutaneous leishmaniasis. The investigational creams were applied topically to all lesions once daily for 20 days. Plasma samples were analyzed for simultaneous quantitation of paromomycin and gentamicin isomers and total gentamicin. Pharmacokinetic parameters for gentamicin could not be calculated because detectable levels were rarely evident. After one application, the paromomycin area under the concentration-time curve from 0 to 24 h (AUC
0–24
) was 2,180 ± 2,621 ng · h/ml (mean ± standard deviation [SD]) for the paromomycin-alone group and 975.6 ± 1,078 ng · h/ml for the WR 279,396 group. After 20 days of application, the paromomycin AUC
0–24
and maximum concentration of drug (
C
max
) were 5 to 6 times greater than those on day 1 for both treatment groups. For the paromomycin-alone group, the AUC
0–24
was 8,575 ± 7,268 ng · h/ml and the
C
max
was 1,000 ± 750 ng/ml, compared with 6,037 ± 3,956 ng · h/ml and 660 ± 486 ng/ml for the WR 279,396 group, respectively. Possibly due to large intersubject variability, no differences (
P
≥ 0.05) in the AUC
0–24
or
C
max
were noted between treatment or between sites on day 1 or 20. The percentage of dose absorbed on day 20 was 12.0% ± 6.26% and 9.68% ± 6.05% for paromomycin alone and WR 279,396, respectively. Paromomycin concentrations in plasma after 20 days of application were 5 to 9% of those after intramuscular administration of 15 mg/kg of body weight/day to adults for the systemic treatment of visceral leishmaniasis. Effective topical treatment of cutaneous leishmaniasis appears to be possible with limited paromomycin and gentamicin systemic absorption, thus avoiding drug accumulation and toxicity. (The work described here has been registered at
ClinicalTrials.gov
under registration no. NCT01032382 and NCT01083576.)
Structures of the Class D Carbapenemases OXA-23 and OXA-146: Mechanistic Basis of Activity against Carbapenems, Extended-Spectrum Cephalosporins, and Aztreonam
Authors: Kaitany Kip-Chumba J. , Klinger Neil V. , June Cynthia M. , Ramey Maddison E. , Bonomo Robert A. , Powers Rachel A. , Leonard David A.
Published: July 2013

Class D β-lactamases that hydrolyze carbapenems such as imipenem and doripenem are a recognized danger to the efficacy of these “last-resort” β-lactam antibiotics. Like all known class D carbapenemases, OXA-23 cannot hydrolyze the expanded-spectrum cephalosporin ceftazidime. OXA-146 is an OXA-23 subfamily clinical variant that differs from the parent enzyme by a single alanine (A220) inserted in the loop connecting β-strands β5 and β6. We discovered that this insertion enables OXA-146 to bind and hydrolyze ceftazidime with an efficiency comparable to those of other extended-spectrum class D β-lactamases. OXA-146 also binds and hydrolyzes aztreonam, cefotaxime, ceftriaxone, and ampicillin with higher efficiency than OXA-23 and preserves activity against doripenem. In this study, we report the X-ray crystal structures of both the OXA-23 and OXA-146 enzymes at 1.6-Å and 1.2-Å resolution. A comparison of the two structures shows that the extra alanine moves a methionine (M221) out of its normal position, where it forms a bridge over the top of the active site. This single amino acid insertion also lengthens the β5-β6 loop, moving the entire backbone of this region further away from the active site. A model of ceftazidime bound in the active site reveals that these two structural alterations are both likely to relieve steric clashes between the bulky R1 side chain of ceftazidime and OXA-23. With activity against all four classes of β-lactam antibiotics, OXA-146 represents an alarming new threat to the treatment of infections caused by
Acinetobacter
spp.
Effect of Pulmonary Surfactant on Antimicrobial Activity
In Vitro
Authors: Schwameis R. , Erdogan-Yildirim Z. , Manafi M. , Zeitlinger M. A. , Strommer S. , Sauermann R.
Published: July 2013

Time-kill curve experiments were performed with linezolid, doripenem, tigecycline, moxifloxacin, and daptomycin against
Staphylococcus aureus
and with colistin, moxifloxacin, and doripenem against
Pseudomonas aeruginosa
to evaluate the effect of porcine pulmonary surfactant on antimicrobial activity. Pulmonary surfactant significantly impaired the activities of moxifloxacin and colistin. When antibiotics are being developed for respiratory tract infections, the method described here might be used to preliminarily quantify the effect of pulmonary surfactant on antimicrobial activity.
Antimicrobial Activity of High-Mobility-Group Box 2: a New Function to a Well-Known Protein
Authors: Küchler Robert , Schroeder Bjoern O. , Jaeger Simon U. , Stange Eduard F. , Wehkamp Jan
Published: July 2013

The human intestinal tract is highly colonized by a vast number of microorganisms. Despite this permanent challenge, infections remain rare, due to a very effective barrier defense system. Essential effectors of this system are antimicrobial peptides and proteins (AMPs), which are secreted by intestinal epithelial and lymphoid cells, balance the gut microbial community, and prevent the translocation of microorganisms. Several antimicrobial proteins have already been identified in the gut. Nonetheless, we hypothesized that additional AMPs are yet to be discovered in this setting. Using biological screening based on antimicrobial function, here we identified competent antibacterial activity of high-mobility-group box 2 (HMGB2) against
Escherichia coli
. By recombinant expression, we confirmed this biologically new antimicrobial activity against different commensal and pathogenic bacteria. In addition, we demonstrated that the two DNA-binding domains (HMG boxes A and B) are crucial for the antibiotic function. We detected HMGB2 in several gastrointestinal tissues by mRNA analysis and immunohistochemical staining. In addition to the nuclei, we also observed HMGB2 in the cytoplasm of intestinal epithelial cells. Furthermore, HMGB2 was detectable
in vitro
in the supernatants of two different cell types, supporting an extracellular function. HMGB2 expression was not changed in inflammatory bowel disease but was detected in certain stool samples of patients, whereas it was absent from control individuals. Taken together, we characterized HMGB2 as an antimicrobial protein in intestinal tissue, complementing the diverse repertoire of gut mucosal defense molecules.
Characterization of the New AmpC β-Lactamase FOX-8 Reveals a Single Mutation, Phe313Leu, Located in the R2 Loop That Affects Ceftazidime Hydrolysis
Authors: Pérez-Llarena Francisco José , Kerff Frédéric , Zamorano Laura , Fernández María Carmen , Nuñez Maria Luz , Miró Elisenda , Oliver Antonio , Navarro Ferran , Bou Germán
Published: July 2013

A novel class C β-lactamase (FOX-8) was isolated from a clinical strain of
Escherichia coli
. The FOX-8 enzyme possessed a unique substitution (Phe313Leu) compared to FOX-3. Isogenic
E. coli
strains carrying FOX-8 showed an 8-fold reduction in resistance to ceftazidime relative to FOX-3. In a kinetic analysis, FOX-8 displayed a 33-fold reduction in
k
cat
/

K
m

for ceftazidime compared to FOX-3. In the FOX family of β-lactamases, the Phe313 residue located in the R2 loop affects ceftazidime hydrolysis and alters the phenotype of
E. coli
strains carrying this variant.
Viral Resistance in Hepatitis C Virus Genotype 1-Infected Patients Receiving the NS3 Protease Inhibitor Faldaprevir (BI 201335) in a Phase 1b Multiple-Rising-Dose Study
Authors: Berger Kristi L. , Lagacé Lisette , Triki Ibtissem , Cartier Mireille , Marquis Martin , Lawetz Carol , Bethell Richard , Scherer Joseph , Kukolj George
Published: July 2013

Faldaprevir (BI 201335) is a selective NS3/4A protease inhibitor under development for the treatment of chronic hepatitis C virus (HCV) infection. NS3/4A genotyping and NS3 protease phenotyping analyses were performed to monitor the emergence of resistance in patients with HCV genotype 1 infection receiving faldaprevir alone or combined with pegylated interferon alfa 2a and ribavirin (PegIFN-RBV) during a phase 1b study. Among all baseline variants, a maximum 7-fold reduction in
in vitro
sensitivity to faldaprevir was observed for a rare NS3 (V/I)170T polymorphism. During faldaprevir monotherapy in treatment-naive patients, virologic breakthrough was common (77%, 20/26) and was associated with the emergence of resistance mutations predominantly carrying NS3 substitutions R155K in GT1a and D168V in GT1b. D168V conferred a greater reduction in faldaprevir sensitivity (1,800-fold) than R155K (330-fold); however, D168V was generally less fit than R155K in the absence of selective drug pressure. Treatment-experienced patients treated with faldaprevir-PegIFN-RBV triple therapy showed higher viral load reductions, lower rates of breakthrough (8%, 5/62), and less frequent emergence of resistance-associated variants compared with faldaprevir monotherapy. (This study has been registered at ClinicalTrials.gov under registration no. NCT00793793.)
Unique Structural Modifications Are Present in the Lipopolysaccharide from Colistin-Resistant Strains of Acinetobacter baumannii
Authors: Pelletier Mark R. , Casella Leila G. , Jones Jace W. , Adams Mark D. , Zurawski Daniel V. , Hazlett Karsten R. O. , Doi Yohei , Ernst Robert K.
Published: July 2013

Acinetobacter baumannii
is a nosocomial opportunistic pathogen that can cause severe infections, including hospital-acquired pneumonia, wound infections, and sepsis. Multidrug-resistant (MDR) strains are prevalent, further complicating patient treatment. Due to the increase in MDR strains, the cationic antimicrobial peptide colistin has been used to treat
A. baumannii
infections. Colistin-resistant strains of
A. baumannii
with alterations to the lipid A component of lipopolysaccharide (LPS) have been reported; specifically, the lipid A structure was shown to be hepta-acylated with a phosphoethanolamine (pEtN) modification present on one of the terminal phosphate residues. Using a tandem mass spectrometry platform, we provide definitive evidence that the lipid A isolated from colistin-resistant
A. baumannii
MAC204 LPS contains a novel structure corresponding to a diphosphoryl hepta-acylated lipid A structure with both pEtN and galactosamine (GalN) modifications. To correlate our structural studies with clinically relevant samples, we characterized colistin-susceptible and -resistant isolates obtained from patients. These results demonstrated that the clinical colistin-resistant isolate had the same pEtN and GalN modifications as those seen in the laboratory-adapted
A. baumannii
strain MAC204. In summary, this work has shown complete structure characterization including the accurate assignment of acylation, phosphorylation, and glycosylation of lipid A from
A. baumannii
, which are important for resistance to colistin.
P2′ Benzene Carboxylic Acid Moiety Is Associated with Decrease in Cellular Uptake: Evaluation of Novel Nonpeptidic HIV-1 Protease Inhibitors Containing P2
bis
-Tetrahydrofuran Moiety
Authors: Yedidi Ravikiran S. , Maeda Kenji , Fyvie W. Sean , Steffey Melinda , Davis David A. , Palmer Ira , Aoki Manabu , Kaufman Joshua D. , Stahl Stephen J. , Garimella Harisha , Das Debananda , Wingfield Paul T. , Ghosh Arun K. , Mitsuya Hiroaki
Published: July 2013

GRL007 and GRL008, two structurally related nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) containing 3(
R
),3a(
S
),6a(
R
)-
bis
-tetrahydrofuranylurethane (
bis
-THF) as the P2 moiety and a sulfonamide isostere consisting of benzene carboxylic acid and benzene carboxamide as the P2′ moiety, respectively, were evaluated for their antiviral activity and interactions with wild-type protease (PR
WT
). Both GRL007 (

K
i

of 12.7 pM with PR
WT
) and GRL008 (

K
i

of 8.9 pM) inhibited PR
WT
with high potency
in vitro
. X-ray crystallographic analysis of PR
WT
in complex with GRL007 or GRL008 showed that the
bis
-THF moiety of both compounds has three direct polar contacts with the backbone amide nitrogen atoms of Asp29 and Asp30 of PR
WT
. The P2′ moiety of both compounds showed one direct contact with the backbone of Asp30′ and a bridging polar contact with Gly48′ through a water molecule. Cell-based antiviral assays showed that GRL007 was inactive (50% effective concentration [EC
50
] of >1 μM) while GRL008 was highly active (EC
50
of 0.04 μM) against wild-type HIV-1. High-performance liquid chromatography (HPLC)/mass spectrometry-based cellular uptake assays showed 8.1- and 84-fold higher intracellular concentrations of GRL008 than GRL007 in human MT-2 and MT-4 cell extracts, respectively. Thus, GRL007, in spite of its favorable enzyme-inhibitory activity and protease binding profile, exhibited a lack of antiviral activity in cell-based assays, most likely due to its compromised cellular uptake associated with its P2′ benzene carboxylic acid moiety. The anti-HIV-1 potency, favorable toxicity, and binding profile of GRL008 suggest that further optimization of the P2′ moiety may improve its antiretroviral features.
Inhibition of Hepatitis C Virus Infection by DNA Aptamer against Envelope Protein
Authors: Yang Darong , Meng Xianghe , Yu Qinqin , Xu Li , Long Ying , Liu Bin , Fang Xiaohong , Zhu Haizhen
Published: July 2013

Hepatitis C virus (HCV) envelope protein (E1E2) is essential for virus binding to host cells. Aptamers have been demonstrated to have strong promising applications in drug development. In the current study, a cDNA fragment encoding the entire E1E2 gene of HCV was cloned. E1E2 protein was expressed and purified. Aptamers for E1E2 were selected by the method of selective evolution of ligands by exponential enrichment (SELEX), and the antiviral actions of the aptamers were examined. The mechanism of their antiviral activity was investigated. The data show that selected aptamers for E1E2 specifically recognize the recombinant E1E2 protein and E1E2 protein from HCV-infected cells. CD81 protein blocks the binding of aptamer E1E2-6 to E1E2 protein. Aptamers against E1E2 inhibit HCV infection in an infectious cell culture system although they have no effect on HCV replication in a replicon cell line. Beta interferon (IFN-β) and IFN-stimulated genes (ISGs) are not induced in virus-infected hepatocytes with aptamer treatment, suggesting that E1E2-specific aptamers do not induce innate immunity. E2 protein is essential for the inhibition of HCV infection by aptamer E1E2-6, and the aptamer binding sites are located in E2. Q412R within E1E2 is the major resistance substitution identified. The data indicate that an aptamer against E1E2 exerts its antiviral effects through inhibition of virus binding to host cells. Aptamers against E1E2 can be used with envelope protein to understand the mechanisms of HCV entry and fusion. The aptamers may hold promise for development as therapeutic drugs for hepatitis C patients.
Population Pharmacokinetics Study of Recommended Zidovudine Doses in HIV-1-Infected Children
Authors: Fauchet Floris , Treluyer Jean-Marc , Frange Pierre , Urien Saik , Foissac Frantz , Bouazza Naim , Benaboud Sihem , Blanche Stephane , Hirt Déborah
Published: July 2013

The aims of this study were to describe the pharmacokinetics of zidovudine (ZDV) and its biotransformation to its metabolite, 3*-azido-3*-deoxy-5*-glucuronylthymidine (G-ZDV), in HIV-infected children, to identify factors that influence the pharmacokinetics of ZDV, and to compare and evaluate the doses recommended by the World Health Organization (WHO) and the Food and Drug Administration (FDA). ZDV concentrations in 782 samples and G-ZDV concentrations in 554 samples from 247 children ranging in age from 0.5 to 18 years were retrospectively measured. A population pharmacokinetic model was developed with NONMEM software (version 6.2), and the pharmacokinetics of ZDV were best described by a one-compartment model with first-order absorption and elimination. The effect of body weight on the apparent elimination clearance and volume of distribution was significant. The mean population parameter estimates were as follows: absorption rate, 2.86 h−1; apparent elimination clearance, 89.7 liters · h−1(between-subject variability, 0.701 liters · h−1); apparent volume of distribution, 229 liters (between-subject variability, 0.807 liters); metabolic formation rate constant, 12.6 h−1(between-subject variability, 0.352 h−1); and elimination rate constant of G-ZDV, 2.27 h−1. On the basis of simulations with FDA and WHO dosing recommendations, the probabilities of observing efficient exposures (doses resulting in exposures of between 3 and 5 mg/liter · h) with less adverse events (doses resulting in exposures below 8.4 mg/liter · h) were higher when the FDA recommendations than when the WHO recommendations were followed. In order to improve the FDA recommendations, ZDV doses should be reconsidered for the weight band (WB) of 20 to 40 kg. The most appropriate doses should be decreased from 9 to 8 mg/kg of body weight twice a day (BID) for the WB from 20 to 29.9 kg and from 300 to 250 mg BID for the WB from 30 to 39.9 kg. The highest dose, 300 mg BID, should be started from body weights of 40 kg.
Globally Expanding Carbapenemase Finally Appears in Spain: Nosocomial Outbreak of Acinetobacter baumannii Producing Plasmid-Encoded OXA-23 in Barcelona, Spain
Authors: Mosqueda Noraida , Espinal Paula , Cosgaya Clara , Viota Sergio , Plasensia Virginia , Álvarez-Lerma Francisco , Montero Milagro , Gómez Julià , Horcajada Juan Pablo , Vila Jordi , Roca Ignasi
Published: July 2013

Resistance of
Acinetobacter baumannii
clinical isolates to carbapenems is on the rise worldwide mainly in association with the production of OXA-23. Until recently, however, OXA-23 was absent in Spain. In this work, we report the molecular characterization of a hospital outbreak of OXA-23-producing
A. baumannii
in Barcelona caused by a multidrug-resistant (MDR) clone belonging to international clone IC-II/sequence type ST85 between October 2010 and May 2011.
bla
OXA-23
was carried in a plasmid of 90 kb and located within the composite transposon Tn
2006
.
Characterization of Hepatitis C Virus Resistance from a Multiple-Dose Clinical Trial of the Novel NS5A Inhibitor GS-5885
Authors: Wong Kelly A. , Worth Angela , Martin Ross , Svarovskaia Evguenia , Brainard Diana M. , Lawitz Eric , Miller Michael D. , Mo Hongmei
Published: July 2013

GS-5885 is a novel hepatitis C virus (HCV) NS5A inhibitor. In a 3-day monotherapy study in treatment-naive genotype 1a (GT1a) and GT1b HCV-infected subjects, median viral load reductions ranged from 2.3 to 3.3 log
10
HCV RNA IU/ml across dosing cohorts (1, 3, 10, 30, or 90 mg once daily). Here, we report viral sequencing and phenotypic analysis of clinical isolates from this study. Detection of baseline NS5A amino acid substitutions at positions 28, 30, 31, or 93 in GT1a was associated with a reduced treatment response. In the GT1b cohort, Y93H was detected in 100% of subjects at day 4 or 14. In the Gt1a cohort, population sequencing detected NS5A resistance-associated mutations at day 4 or 14 for 3/10 subjects at the 1-mg dose and for all subjects dosed at ≥3 mg. A subset of mutants that confer a low level of reduced susceptibility to GS-5885 was not detected by population sequencing at the 30- and 90-mg doses. Subject-derived M28T, Q30R, L31M, and Y93C mutations all conferred >30-fold reductions in GS-5885 and daclatasvir susceptibilities
in vitro
. Site-directed NS5A mutants also showed reduced susceptibility to GS-5885. However, all NS5A mutants tested remained fully susceptible to other classes of direct-acting antivirals (DAAs), interferon alpha, and ribavirin. Importantly, the nonoverlapping resistance profile and high potency of GS-5885 support its further development with other direct-acting antivirals for the treatment of chronic HCV. (This study has been registered at ClinicalTrials.gov under registration number NCT01193478.)