Multiple hydrogen-bonded dimers: are only the frontier atoms relevant?

Non-frontier atom exchanges in hydrogen-bonded aromatic dimers can induce significant interaction energy changes (up to 6.5 kcal mol-1). Our quantum-chemical analyses reveal that the relative hydrogen-bond strengths of N-edited guanine-cytosine base pair isosteres, which cannot be explained from the frontier atoms, follow from the charge accumulation in the monomers.

Lessons for Oral Bioavailability: How Conformationally Flexible Cyclic Peptides Enter and Cross Lipid Membranes.

Cyclic peptides extend the druggable target space due to their size, flexibility, and hydrogen-bonding capacity. However, these properties impact also their passive membrane permeability. As the "journey" through membranes cannot be monitored experimentally, little is known about the underlying process, which hinders rational design. Here, we use molecular simulations to uncover how cyclic peptides permeate a membrane. We show that side chains can act as "molecular anchors", establishing the first contact with the membrane and enabling insertion. Once inside, the peptides are positioned between headgroups and lipid tails─a unique polar/apolar interface. Only one of two distinct orientations at this interface allows for the formation of the permeable "closed" conformation. In the closed conformation, the peptide crosses to the lower leaflet via another "anchoring" and flipping mechanism. Our findings provide atomistic insights into the permeation process of flexible cyclic peptides and reveal design considerations for each step of the process.

Combined treatment of Nimotuzumab and rapamycin is effective against temozolomide-resistant human gliomas regardless of the EGFR mutation status.

BACKGROUND: The treatment of glioblastoma multiforme (GBM) is an unmet clinical need. The 5-year survival rate of patients with GBM is less than 3%. Temozolomide (TMZ) remains the standard first-line treatment regimen for gliomas despite the fact that more than 90% of recurrent gliomas do not respond to TMZ after repeated exposure. We have also independently shown that many of the Asian-derived glioma cell lines and primary cells derived from Singaporean high-grade glioma patients are indeed resistant to TMZ. This issue highlights the need to develop new effective anti-cancer treatment strategies. In a recent study, wild-type epidermal growth factor receptor (wtEGFR) has been shown to phosphorylate a truncated EGFR (known as EGFRvIII), leading to the phosphorylation of STAT proteins and progression in gliomagenesis. Despite the fact that combination of EGFR targeting drugs and rapamycin has been used before, the effect of mono-treatment of Nimotuzumab, rapamycin and combination therapy in human glioma expressing different types of EGFR is not well-studied. Herein, we evaluated the efficacy of dual blockage using monoclonal antibody against EGFR (Nimotuzumab) and an mTOR inhibitor (rapamycin) in Caucasian patient-derived human glioma cell lines, Asian patient-derived human glioma cell lines, primary glioma cells derived from the Mayo GBM xenografts, and primary short-term glioma culture derived from high-grade glioma patients. METHODS: The combination effect of Nimotuzumab and rapamycin was examined in a series of primary human glioma cell lines and glioma cell lines. The cell viability was compared to TMZ treatment alone. Endogenous expressions of EGFR in various GBM cells were determined by western blotting. RESULTS: The results showed that combination of Nimotuzumab with rapamycin significantly enhanced the therapeutic efficacy of human glioma cells compared to single treatment. More importantly, many of the Asian patient-derived glioma cell lines and primary cells derived from Singaporean high-grade gliomas, which showed resistance to TMZ, were susceptible to the combined treatments. CONCLUSIONS: In conclusion, our results strongly suggest that combination usage of Nimotuzumab and rapamycin exert higher cytotoxic activities than TMZ. Our data suggest that this combination may provide an alternative treatment for TMZ-resistant gliomas regardless of the EGFR status.

3D imaging of cement-based materials at submicron resolution by combining laser scanning confocal microscopy with serial sectioning.

In this paper, we present a new method to reconstruct large volumes of nontransparent porous materials at submicron resolution. The proposed method combines fluorescence laser scanning confocal microscopy with serial sectioning to produce a series of overlapping confocal z-stacks, which are then aligned and stitched based on phase correlation. The method can be extended in the XY plane to further increase the overall image volume. Resolution of the reconstructed image volume does not degrade with increase in sample size. We have used the method to image cementitious materials, hardened cement paste and concrete and the results obtained show that the method is reliable. Possible applications of the method such as three-dimensional characterization of the pores and microcracks in hardened concrete, three-dimensional particle shape characterization of cementitious materials and three-dimensional characterization of other porous materials such as rocks and bioceramics are discussed.

Utility of the sendai consensus guidelines for branch-duct intraductal papillary mucinous neoplasms: a systematic review.

INTRODUCTION: The Sendai Consensus Guidelines (SCG) was formulated in 2006 to guide the management of intraductal papillary mucinous neoplasms (IPMN). The main area of controversy is the criteria for selection of branch duct (BD)-IPMN for resection. Although these guidelines have gained widespread acceptance, there is limited data to date supporting its use. This systematic review is performed to evaluate the utility of the Sendai Consensus Guidelines (SCG) for BD-IPMN. METHODS: Studies evaluating the clinical utility of the SCG in surgically resected neoplasms were identified. The SCG were retrospectively applied to all resected neoplasms in these studies. BD-IPMNs which met the criteria for resection were termed SCG+ve and those for surveillance were termed SCG-ve. RESULTS: Twelve studies were included, of which, 9 were suitable for pooled analysis. There were 690 surgically resected BD-IPMNs, of which, 24% were malignant. Five hundred one BD-IPMNs were classified as SCG+ve and 189 were SCG-ve. The positive predictive value (PPV) of SCG+ve neoplasms ranged from 11 to 52% and the NPV of SCG-ve neoplasms ranged from 90 to 100%. Overall, there were 150/501 (29.9%) of malignant BD-IPMNs in the SCG+ve group and 171/189 (90%) of benign BD-IPMNs in the SCG-ve group. Of the 18 reported malignant (11 invasive) BD-IPMNs in the SCG-ve group, 17 (including all 11 invasive) were from a single study. When the results from this single study were excluded, 170/171 (99%) of SCG-ve BD-IPMNs were benign. CONCLUSION: The results of this review confirm the limitations of the SCG for BD-IPMN. The PPV of the SCG in predicting a malignant BD-IPMN was low and some malignant lesions may be missed based on these guidelines.

Transport of L-carnitine in isolated cerebral cortex neurons.

The accumulation of carnitine was measured in cerebral cortex neurons isolated from adult rat brain. This process was found to be lowered by 40% after preincubation with ouabain and with SH-group reagents (N-ethylmaleimide and mersalyl). The initial velocity of carnitine transport was found to be inhibited by 4-aminobutyrate (GABA) in a competitive way (Ki = 20.9 +/- 2.4 mM). However, of various inhibitors of GABA transporters, only nipecotic acid and very high concentrations of 1-[2-([(diphenylmethylene)amino]oxy)ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride (NO-711) acid decreased carnitine accumulation while betaine, taurine and beta-alanine had no effect. The GABA transporters expressed in Xenopus laevis oocytes did not transport carnitine. Moreover, carnitine was not observed to diminish the accumulation of GABA in cerebral cortex neurons, which further excluded a possible involvement of the GABA transporter GAT1 in the process of carnitine accumulation, despite the expression of this protein in the cells under study. The absence of carnitine transporter OCTN2 in rat cerebral cortex neurons (K. A. Nalecz, D. Dymna, J. E. Mroczkowska, A. Broër, S. Broër, M. J. Nalecz and R. Cecchelli, unpublished results), together with the insensitivity of carnitine accumulation towards betaines, implies that a novel transporting protein is present in these cells.

Transport of alpha-ketoisocaproate in rat cerebral cortical neurons.

Transport of alpha-ketoisocaproic acid (KIC), the product of leucine transamination, was studied in the cerebral cortex cells isolated from the adult rat brain. The process of [(14)C]KIC accumulation was followed in the presence of aminooxyacetate, an inhibitor of transaminases. Accumulation of KIC was not affected by Na(+) replacement, its initial velocity was observed to be higher upon lowering of external pH. Addition of KIC promoted acidification of cytoplasmic pH, monitored with 2'7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. The detected inhibition of KIC accumulation by alpha-cyano-4(OH)cinnamate pointed to an involvement of one of monocarboxylate transporters (MCT), although 4,4'-diisothiocyano-2,2'-stilbenedisulphonate was without effect. Accumulation of KIC was inhibited by lactate; the effect of pyruvate was detected to be much weaker. Other branched-chain alpha-ketoacids (ketoisovalerate, keto-methylvalerate), as well as beta-hydroxybutyrate and valproate decreased the transport of KIC by 30, 60, and 80%, respectively. The observed characteristics of KIC accumulation in the cortical neurons indicate an involvement of one of the MCT transporters. A crucial role of SH group(s) in the process of KIC accumulation, excluding MCT2, indicates the MCT1, although an involvement of another isoform of MCT in the process of KIC transport in neurons from cerebral cortex of adult brain has not been definitely excluded.

Evaluation of an aqueous drainage glaucoma device constructed of ePTFE.

Aqueous drainage devices for the treatment of glaucoma are subject to the same limitations as most polymeric implants, namely a healing response comprised of chronic inflammation and fibrosis. The most widely used devices are currently made of silicone or polypropylene, materials that exhibit biocompatibility difficulties when they are implanted on the sclera underneath the conjunctiva of the eye. Decreased outflow of aqueous fluid to the conjunctival space caused by the development of a fibrous capsule around the device accounts for at least 20% of aqueous shunts failures. Clearly, the need exists to improve the healing response to aqueous drainage devices, and one approach is to develop new polymers or polymer modifications. Improved devices would elicit a limited fibrotic response while increasing neovascularization around the implant. Previous studies have indicated that denucleation markedly improves the healing characteristics and biocompatibility of expanded polytetrafluoroethylene (ePTFE). We reasoned that altering the design of drainage devices to allow the use of denucleated ePTFE in vivo might minimize fibrosis, thereby improving shunt function. We found that after 8 weeks in vivo, experimental shunt function was equivalent to the Baerveldt shunt, while there was less scarring with increased neovascularizatin. These findings suggest that ePTFE has potential as an improved, long-term alternative material for use in constructing glaucoma shunts.

The U.S. federal framework for research on endocrine disruptors and an analysis of research programs supported during fiscal year 1996.

The potential health and ecological effects of endocrine disrupting chemicals has become a high visibility environmental issue. The 1990s have witnessed a growing concern, both on the part of the scientific community and the public, that environmental chemicals may be causing widespread effects in humans and in a variety of fish and wildlife species. This growing concern led the Committee on the Environment and Natural Resources (CENR) of the National Science and Technology Council to identify the endocrine disruptor issue as a major research initiative in early 1995 and subsequently establish an ad hoc Working Group on Endocrine Disruptors. The objectives of the working group are to 1) develop a planning framework for federal research related to human and ecological health effects of endocrine disrupting chemicals; 2) conduct an inventory of ongoing federal research programs; and 3) identify research gaps and develop a coordinated interagency plan to address priority research needs. This communication summarizes the activities of the federal government in defining a common framework for planning an endocrine disruptor research program and in assessing the status of the current effort. After developing the research framework and compiling an inventory of active research projects supported by the federal government in fiscal year 1996, the CENR working group evaluated the current federal effort by comparing the ongoing activities with the research needs identified in the framework. The analysis showed that the federal government supports considerable research on human health effects, ecological effects, and exposure assessment, with a predominance of activity occurring under human health effects. The analysis also indicates that studies on reproductive development and carcinogenesis are more prevalent than studies on neurotoxicity and immunotoxicity, that mammals (mostly laboratory animals) are the main species under study, and that chlorinated dibenzodioxins and polychlorinated biphenyls are the most commonly studied chemical classes. Comparison of the inventory with the research needs should allow identification of underrepresented research areas in need of attention.

Transport of alpha-ketoisocaproate in neuroblastoma NB-2a cells.

Transport of alpha-ketoisocaproate (KIC), a ketoacid originating from leucine and proposed to be involved in the buffering of glutamate in neurones, was studied in neuroblastoma NB-2a cells. The accumulated KIC was mostly transaminated to leucine, while free keto-acid was detectable either only after prolonged times or after inhibiting transaminase with aminooxyacetate. Accumulation of KIC was found to be inhibited by other branched-chain ketoacids, while lactate and beta-hydroxybutyrate were ineffective. The transport of KIC, resembling a facilitated diffusion, was decreased by phloretin, alpha-cyano-4-hydroxycinnamate, 4,4'-diisothiocyano-2,2'-stilbenedisulphonate, and p-chlorimercuribenzoate. The process of accumulation did not resemble a symport with protons; therefore an involvement of the known proton-coupled monocarboxylate transporters (MCT) was excluded. Distribution of KIC suggests a mechanism involving a cotransport with 2 [Na+].

Identification of histidine as an axial ligand to P700+.

The primary electron donor in photosystem I (PSI), P700, is thought to be a dimeric Chl a species. Neither the electronic nor geometric structure of the cation radical is clearly understood. Magnetic resonance studies have indicated that the unpaired electron in P700+ is delocalized asymmetrically over the Chl dimer; however, the axial ligand to the central Mg2+ is not known. The recent development of a histidine tolerant mutant of Synechocystis PCC 6803 has allowed us to use a combination of isotopic labeling and electron nuclear double resonance (ENDOR) spectroscopy to show the first definitive spectroscopic evidence of a histidine ligand to P700+. Peaks split symmetrically about the 15N Larmor frequency corresponding to an isotropic hyperfine coupling of 0.64 MHz were observed in the ENDOR spectra from P700+ globally labeled with 15N and specifically labeled with [15N]histidine. These peaks disappeared in "reverse" labeled samples in which all nitrogens are 15N except those of histidine, which contains natural abundance 14N. The dipolar contribution to the hyperfine coupling was determined by using electron spin echo envelope modulation spectroscopy (ESEEM). Numerical simulations of the ESEEM data suggest that the coupling is primarily isotropic and that the histidine is directly coordinated to the central Mg2+ of P700+. Taken together, these data are supportive of a model of P700+ in which the excited state molecular orbital makes a significant contribution to the electronic structure of the radical. Moreover, the methodology developed in this work can be extended to examine the magnetic properties of axial ligands in a variety of biologically relevant porphyrin/chlorin systems.

Research needs for the risk assessment of health and environmental effects of endocrine disruptors: a report of the U.S. EPA-sponsored workshop.

The hypothesis has been put forward that humans and wildlife species adverse suffered adverse health effects after exposure to endocrine-disrupting chemicals. Reported adverse effects include declines in populations, increases in cancers, and reduced reproductive function. The U.S. Environmental Protection Agency sponsored a workshop in April 1995 to bring together interested parties in an effort to identify research gaps related to this hypothesis and to establish priorities for future research activities. Approximately 90 invited participants were organized into work groups developed around the principal reported health effects-carcinogenesis, reproductive toxicity, neurotoxicity, and immunotoxicity-as well as along the risk assessment paradigm-hazard identification, dose-response assessment, exposure assessment, and risk characterization. Attention focused on both ecological and human health effects. In general, group felt that the hypothesis warranted a concerted research effort to evaluate its validity and that research should focus primarily on effects on development of reproductive capability, on improved exposure assessment, and on the effects of mixtures. This report summarizes the discussions of the work groups and details the recommendations for additional research.

Environmental contaminants and the reproductive success of lake trout in the Great Lakes: an epidemiological approach.

Epidemiological criteria were used to examine the influence of environmental contamination on reproductive success of lake trout (Salvelinus namaycush) in the Laurentian Great Lakes. Most of the information was obtained from lake trout eggs collected in southeastern Lake Michigan and reared in the laboratory. Two separate end points that measure reproductive success--egg hatchability and fry survival--were used in the evaluation. Strong evidence for maternally derived polychlorinated biphenyls causing reduced egg hatchability were observed for the time order, strength of association, and coherence criteria. Equally strong evidence for organic environmental contaminants, also of maternal origin, causing a swim-up fry mortality syndrome were presented for the strength of association, specificity, replication, and coherence criteria. The epidemiological approach for demonstrating cause-and-effect relations was useful because of the difficulty in demonstrating definite proof of causality between specific environmental contaminants and reproductive dysfunction in feral fish.