Mechanism of lipid bilayer perturbation by bactericidal membrane-active small molecules.

Membrane-active small molecules (MASMs) are small organic molecules designed to reproduce the fundamental physicochemical properties of natural antimicrobial peptides: their cationic charge and amphiphilic character. This class of compounds has a promising broad range of antimicrobial activity and, at the same time, solves some major limitations of the peptides, such as their high production costs and low in vivo stability. Most cationic antimicrobial peptides act by accumulating on the surface of bacterial membranes and causing the formation of defects when a threshold is reached. Due to the drastically different structures of the two classes of molecules, it is not obvious that small-molecule antimicrobials act in the same way as natural peptides, and very few data are available on this aspect. Here we combined spectroscopic studies and molecular dynamics simulations to characterize the mechanism of action of two different MASMs. Our results show that, notwithstanding their simple structure, these molecules act just like antimicrobial peptides. They bind to the membrane surface, below the head-groups, and insert their apolar moieties in the core of the bilayer. Like many natural peptides, they cause the formation of defects when they reach a high coverage of the membrane surface. In addition, they cause membrane aggregation, and this property could contribute to their antimicrobial activity.

Analysis of Antibacterial Action of Mammalian Host-Defense Cathelicidins and Induction of Resistance to Them in MßL-Producing Pseudomonas aeruginosa.

Recombinant analogs of a number of natural host-defense mammalian cathelicidins were obtained and predominant mechanism of their antibacterial action was studied. The ability of cathelicidins to suppress the growth of Pseudomonas aeruginosa producing metallo-ß-lactamases (MßL) was studied, and the possibility of appearance of cathelicidin-resistant bacteria was evaluated. Among peptides with different structures and mechanisms of action, only the strains resistant to ChMAP-28 were not obtained, which indicated minimum risk of the development of natural resistance to this cathelicidin. High antibacterial activity, wide spectrum of action, and the absence of cross-resistance effects allow considering ChMAP-28 peptide as a candidate to be developed further as a therapeutic agent against MßL-producing bacteria.

Individual parcellation of resting fMRI with a group functional connectivity prior.

Cortical parcellation based on resting fMRI is an important tool for investigating the functional organization and connectivity of the cerebral cortex. Group parcellation based on co-registration of anatomical images to a common atlas will inevitably result in errors in the locations of the boundaries of functional parcels when they are mapped back from the atlas to the individual. This is because areas of functional specialization vary across individuals in a manner that cannot be fully determined from the sulcal and gyral anatomy that is used for mapping between atlas and individual. We describe a method that avoids this problem by refining an initial group parcellation so that for each subject the parcel boundaries are optimized with respect to that subject's resting fMRI. Initialization with a common parcellation results in automatic correspondence between parcels across subjects. Further, by using a group sparsity constraint to model connectivity, we exploit group similarities in connectivity between parcels while optimizing their boundaries for each individual. We applied this approach with initialization on both high and low density group cortical parcellations and used resting fMRI data to refine across a group of individuals. Cross validation studies show improved homogeneity of resting activity within the refined parcels. Comparisons with task-based localizers show consistent reduction of variance of statistical parametric maps within the refined parcels relative to the group-based initialization indicating improved delineation of regions of functional specialization. This method enables a more accurate estimation of individual subject functional areas, facilitating group analysis of functional connectivity, while maintaining consistency across individuals with a standardized topological atlas.

Use of an Innovative Simple Method for Anaerobiosis in the Diagnosis and Management of Infections in Two Unusual Cases.

Technical limitations restrict routine anaerobe isolation from clinical materials in resource-limited laboratories. An innovative two steps combustion candle jar technique may be suitable for such setup. This system was tried with one case of chronic osteomyelitis developed on supracondyler compound fracture. Porphyromonas spp. was isolated and identified. Vancomycin was recommended based on in vitro sensitivity test, but the leg was amputed after receiving a resistant drug gentamycin. While in another child with hydrocephalous, V-P shunt associated infection by Peptostreptococcus anaerobius was successfully controlled by sensitive drug vancomycin. These two eye-opener cases insisted us for large scale application of the technique.

Cyclization and unsaturation rather than isomerisation of side chains govern the selective antibacterial activity of cationic-amphiphilic polymers.

Membrane-active agents represent a promising alternative to overcome antibiotic resistance. Here, we report cationic-amphiphilic polymers with variations in the side chain architecture such as cyclization, isomerization and unsaturation that resulted in potent antibacterial activity and low mammalian cell toxicity with a membrane-active mode of action.

Isosteric substitution in cationic-amphiphilic polymers reveals an important role for hydrogen bonding in bacterial membrane interactions.

Biomimetic antibacterial polymers, the functional mimics of antimicrobial peptides (AMPs), targeting the bacterial cell membrane have been developed to combat the problem of antibiotic resistance. Amphiphilicity, a balance of cationic charge and hydrophobicity, in these polymers has been shown to be pivotal for their selective interactions with anionic lipid membranes of bacteria instead of zwitterionic mammalian (human erythrocyte) membranes. However, it is unclear if and to what extent hydrogen bonding in amphiphilic antibacterial polymers contributes to this membrane binding specificity. To address this, we employ isosteric substitution of ester with amide moieties that differ in their potency for hydrogen bonding in the side chains of N-alkyl maleimide based amphiphilic polymers. Our studies reveal that amide polymer (AC3P) is a potent antibacterial agent with high membrane-disrupting properties compared to its ester counterpart (EC3P). To understand these differences we performed bio-physical experiments and molecular dynamics (MD) simulations which showed strong interactions of AC3P including hydrogen bonding with lipid head groups of bacterial model lipid bilayers, that are absent in EC3P, make them selective for bacterial membranes. Mechanistic investigations of these polymers in bacteria revealed specific membrane disruptive activity leading to the delocalization of cell division related proteins. This unprecedented and unique concept provides an understanding of bacterial membrane interactions highlighting the role of hydrogen bonding. Thus, these findings will have significant implications in efficient design of potent membrane-active agents.

Anaerobic culture on growth efficient bi-layered culture plate in a modified candle jar using a rapid and slow combustion system.

Success for maximum isolation of anaerobes depends on maintaining critically low oxygen levels throughout and growth in a reduced medium with exclusion of inhibitory substances. Hence a dual system was used equipped with candle combustion for instant exhaustion of major part of oxygen from a sealed jar, along with acidified steel wool for residual oxygen purging. For inhibitory substances removal, test anaerobes were grown on anaerobic medium layered on buffer charcoal agar bed. After 48 hours incubation average colony sizes were compared with that of growths in conventional Gas-Pak system. Better growths were noted in the innovative system.

Removal of olefinic fat chemical shift artifact in diffusion MRI.

Diffusion-weighted (DW) MRI has emerged as a key tool for assessing the microstructure of tissues in healthy and diseased states. Because of its rapid acquisition speed and insensitivity to motion, single-shot echo-planar imaging is the most common DW imaging technique. However, the presence of fat signal can severely affect DW-echo planar imaging acquisitions because of the chemical shift artifact. Fat suppression is usually achieved through some form of chemical shift-based fat saturation. Such methods effectively suppress the signal originating from aliphatic fat protons, but fail to suppress the signal from olefinic protons. Olefinic fat signal may result in significant distortions in the DW images, which bias the subsequently estimated diffusion parameters. This article introduces a method for removing olefinic fat signal from DW images, based on an echo time-shifted acquisition. The method is developed and analyzed specifically in the context of single-shot DW-echo-planar imaging, where image phase is generally unreliable. The proposed method is tested with phantom and in vivo datasets, and compared with a standard acquisition to demonstrate its performance.

Robust water/fat separation in the presence of large field inhomogeneities using a graph cut algorithm.

Water/fat separation is a classical problem for in vivo proton MRI. Although many methods have been proposed to address this problem, robust water/fat separation remains a challenge, especially in the presence of large amplitude of static field inhomogeneities. This problem is challenging because of the nonuniqueness of the solution for an isolated voxel. This paper tackles the problem using a statistically motivated formulation that jointly estimates the complete field map and the entire water/fat images. This formulation results in a difficult optimization problem that is solved effectively using a novel graph cut algorithm, based on an iterative process where all voxels are updated simultaneously. The proposed method has good theoretical properties, as well as an efficient implementation. Simulations and in vivo results are shown to highlight the properties of the proposed method and compare it to previous approaches. Twenty-five cardiac datasets acquired on a short, wide-bore scanner with different slice orientations were used to test the proposed method, which produced robust water/fat separation for these challenging datasets. This paper also shows example applications of the proposed method, such as the characterization of intramyocardial fat.

Joint estimation of water/fat images and field inhomogeneity map.

Water/fat separation in the presence of B 0 field inhomogeneity is a problem of considerable practical importance in MRI. This article describes two complementary methods for estimating the water/fat images and the field inhomogeneity map from Dixon-type acquisitions. One is based on variable projection (VARPRO) and the other on linear prediction (LP). The VARPRO method is very robust and can be used in low signal-to-noise ratio conditions because of its ability to achieve the maximum-likelihood solution. The LP method is computationally more efficient, and is shown to perform well under moderate levels of noise and field inhomogeneity. These methods have been extended to handle multicoil acquisitions by jointly solving the estimation problem for all the coils. Both methods are analyzed and compared and results from several experiments are included to demonstrate their performance.

Accelerating Advanced MRI Reconstructions on GPUs.

Computational acceleration on graphics processing units (GPUs) can make advanced magnetic resonance imaging (MRI) reconstruction algorithms attractive in clinical settings, thereby improving the quality of MR images across a broad spectrum of applications. This paper describes the acceleration of such an algorithm on NVIDIA's Quadro FX 5600. The reconstruction of a 3D image with 128(3) voxels achieves up to 180 GFLOPS and requires just over one minute on the Quadro, while reconstruction on a quad-core CPU is twenty-one times slower. Furthermore, relative to the true image, the error exhibited by the advanced reconstruction is only 12%, while conventional reconstruction techniques incur error of 42%.

A network flow method for improved MR field map estimation in the presence of water and fat.

Field map estimation is an important problem in MRI, with applications such as water/fat separation and correction of fast acquisitions. However, it constitutes a nonlinear and severely ill-posed problem requiring regularization. In this paper, we introduce an improved method for regularized field map estimation, based on a statistically motivated formulation, as well as a novel algorithm for the solution of the corresponding optimization problem using a network flow approach. The proposed method provides theoretical guarantees (local optimality with respect to a large move), as well as an efficient implementation. It has been applied to the water/fat separation problem and tested on a number of challenging datasets, showing high-quality results.

Prevalence of bancroftian filariasis in Burdwan district, West Bengal: II. Vector and microfilariae density in colliery and non-colliery areas.

The paper reports the density of Culex quinquefasciatus, infection and infectivity rates, microfilariae(mf)- density and microfilaraemic persons in the colliery and non-colliery areas of Burdwan district, West Bengal. The numbers C. quinquefasciatus were significantly higher in colliery areas, as compared to non-colliery areas. The infection rate and infectivity rates of the vector were found to be higher in colliery areas. The higher vector density in colliery area is one of the major reasons of higher prevalence of filariasis in that area. However, other environmental factors like exposure of the host to the coal might influence the pathogenesis of filariasis in the colliery area.

DNA base excision repair of 5-hydroxymethyl-2'-deoxyuridine stimulates poly(ADP-ribose) synthesis in Chinese hamster cells.

5-Hydroxymethyl-2'-deoxyuridine (hmdUrd) is incorporated into DNA as a thymidine analog resulting in extensive substitution of thymine residues with 5-hydroxymethyluracil (hmUra) residues. These hmUra residues are then subject to excision by action of hmUra-DNA glycosylase. 3-Aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) synthesis, is toxic to cells that incorporate and repair hmdUrd. To demonstrate that incorporation and repair of hmdUrd stimulates synthesis of poly(ADP-ribose) from intracellular NAD, V79 hamster cells were treated with hmdUrd and intracellular NAD levels were measured. Following hmdUrd treatment, NAD levels fell markedly (80-90%) within 4 h and remained low for at least 10 h, before partially recovering by 24 h. The degree of NAD lowering was dose dependent and paralleled net hmdUrd incorporation. The NAD lowering was largely prevented by concurrent treatment with 4 mM 3AB. No effects on NAD levels were seen following treatment with deoxythymidine or bromodeoxyuridine, which are incorporated into DNA but, in contrast to hmdUrd, are not repaired. When the incorporation of hmdUrd into DNA was blocked with hydroxyurea or aphidicolin, no NAD lowering was seen. HmdUrd also did not produce lowering of NAD concentrations in mutant cell strains deficient in the ability either to incorporate hmdUrd into DNA or to repair hmdUrd from DNA. These results demonstrate that synthesis of poly(ADP-ribose) resulted directly from the incorporation into DNA of the nucleoside hmdUrd and its subsequent repair. These results unequivocally demonstrate that the initiation of normal DNA base excision repair by itself, and not DNA damage per se, is a sufficient stimulus for the induction of poly(ADP-ribose) synthesis.

Opioid modulation of oxytocin release from spinal cord synaptosomes.

Regulation of oxytocin release from spinal cord synaptosomes was investigated using in vitro preparations. Experiments were designed to determine whether opioid peptides regulate oxytocin release from spinal cord synaptosomes, as they do from synaptosomes derived from neurohypophysis. Oxytocin release was evoked by the addition of 56 mM KCl in synaptosomes prepared from thoracic and lumbosacral parts of the spinal cord. Addition of 5 microM naloxone, 1 min prior to the addition of the stimulus, caused a significant (p < 0.025) increase of oxytocin release. Prior addition of 5 microM dynorphin, demonstrated a significant (p < 0.01) decrease whereas addition of 5 microM [D-Ala2,N-Me-Phe4,Gly-ol]-enkephalin showed no effect on KCl-induced OT release. The results suggest that spinal cord OT release is under inhibitory control of opioid peptides and the opioids act via the kappa opioid receptor.

Prevalence of Bancroftian filariasis in Burdwan District, west Bengal: a comparative study between colliery and non-colliery areas.

The present study reports results of a survey of bancroftian filariasis in Burdwan district West Bengal. 85,974 persons were examined of which 46,221 were inhabitants of the colliery areas of the district and rest 39,753 were from non-Colliery areas, including urban and rural areas of the district. The clinicopositivity, endemicity, microfilaria(mf)-positivity, mf-clinicopositivity, and mf-density were determined. It was found that all these epidemiological and parasitological parameters were significantly higher in the colliery area as compared to non-colliery area. The high vector density in the colliery area may account for such significantly higher prevalence of filariasis in this area. From a differential count of neutrophils, eosinophils and lymphocytes in the mf-positive cases, it was revealed that neutrophil percentage was significantly (P < 0.01) decreased while eosinophils and Lymphocytes increased. The decrease of neutrophils was correlated ((Correlation co-efficient, r = -0.78) with the increase of lymphocytes, indicating an imbalance in the immune-system of the infected persons. The geographical and geological factors resulting in high vector density are considered responsible for the significantly higher rate of bancroftian filariasis in the Colliery area.

The release of oxytocin from spinal cord synaptosomes by high KCl depolarizing stimulus: a calcium dependent process.

Oxytocin (OT) release from synaptosomes isolated from the thoracic (T) and lumbosacral (LS) regions of the spinal cord was evoked by 56 mM potassium chloride (KCl). The release mechanism was shown to be a calcium dependent process. The ability of high KCl to evoke OT release from isolated nerve terminals in a calcium dependent manner provides additional support for the role of OT as a neurotransmitter in the spinal cord.

Isotonic and hypertonic saline act as stressful stimuli for oxytocinergic system of the pituitary, hypothalamus and spinal cord.

We have recently demonstrated that immobilization stress leads to an increase in the spinal cord oxytocin content in the rat. The current experiments were undertaken to determine if other stressors have similar effect on the spinal cord oxytocin levels. Male Long Evans rats were injected either with isotonic or hypertonic saline and sacrificed either 15 minutes or 3 hours after saline injection. Oxytocin content of the neurohypophysis, hypothalamus and spinal cord were determined by specific radioimmunoassay in Sep-pak extracted samples. The results demonstrate that both isotonic and hypertonic saline act as stressful stimuli and reduce oxytocin content of the pituitary and hypothalamus when the rats were sacrificed within 15 minute following the injection. Spinal cord oxytocin content was also affected by isotonic and hypertonic saline administration; oxytocin content decreased if rats were sacrificed after a short period (15 min) and increased if rats were sacrificed after a long period (3 hours). These results, together with those reported earlier, support the hypothesis that stressors, in general, affect the spinal cord oxytocin content.