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Abstract cGMP-dependent protein kinase I-α (PKG1α) is a target for pulmonary arterial hypertension due to its role in the regulation of smooth muscle function. While most work has focused on regulation of cGMP turnover, we recently described several small molecule tool compounds which were capable of activating PKG1α via a cGMP independent pathway. Selected molecules were crystallized in the presence of PKG1α and were found to bind to an allosteric site proximal to the low-affinity nucleotide binding domain. These molecules act to displace the switch helix and cause activation of PKG1α representing a new mechanism for the activation and control of this critical therapeutic path. The described structures are vital to understanding the function and control of this key regulatory pathway.

A novel exonuclease, designated as MszExo I, was cloned from Methylocaldum szegediense, a moderately thermophilic methanotroph. It specifically digests single-stranded DNA in the 3' to 5' direction. The protein is composed of 479 amino acids, and it shares 47% sequence identity with E. coli Exo I. The crystal structure of MszExo I was determined to a resolution of 2.2 Å and it aligns well with that of E. coli Exo I. Comparative studies revealed that MszExo I and E. coli Exo I have similar metal ion binding affinity and similar activity at mesophilic temperatures (25-47°C). However, the optimum working temperature of MszExo I is 10°C higher, and the melting temperature is more than 4°C higher as evaluated by both thermal inactivation assays and DSC measurements. More importantly, two thermal transitions during unfolding of MszExo I were monitored by DSC while only one transition was found in E. coli Exo I. Further analyses showed that magnesium ions not only confer structural stability, but also affect the unfolding of MszExo I. MszExo I is the first reported enzyme in the DNA repair systems of moderately thermophilic bacteria, which are predicted to have more efficient DNA repair systems than mesophilic ones.

Inhibition of leucine-rich repeat kinase 2 is a genetically supported mechanism for the treatment of Parkinson's disease. We previously disclosed the discovery of an indazole series lead that demonstrated both safety and translational risks. The safety risks were hypothesized to be of unknown origin, so structural diversity in subsequent chemical matter was prioritized. The translational risks were identified due to a low brain Kp u,u in nonhuman primate studies, which raised concern over the use of an established peripheral biomarker as a surrogate for central target engagement. Given these challenges, the team sought to leverage structure- and property-based drug design and expanded efflux transporter profiling to identify structurally distinct leads with enhanced CNS drug-likeness. Herein, we describe the discovery of a "reinvented" indazole series with improved physicochemical properties and efflux transporter profiles while maintaining excellent potency and off-target kinase selectivity, which resulted in advanced lead, compound 23 .

A fill-and-draw flushing test on a landfill cell containing MSW waste was carried out to examine the operational viability of this method for accelerating the flushing of contaminants and landfill stabilisation. During the fill cycle, 800 m 3 of water containing the tracer bromide was pumped into the base of a 0.44 ha landfill cell, resulting in the estimated saturation of 9,400 m 3 of waste. Abstraction took place in two phases, during which 1,100 m 3 of tracer/leachate was recovered. Samples of leachate were analysed for the tracer, electrical conductivity and indigenous solutes chloride and ammonia. Tracer recovery was between 63 and 72 % for bromide. An estimated 227 kg of ammonia and 575 kg of chloride were removed. Test data was used to calibrate a 1D, dual-porosity model involving advection in a mobile zone, and diffusion into 'blocks' of a less mobile zone. The model fitted well to the early time data, whereas later data appears to have been affected by recharge. The results of this trial demonstrate the possibilities of the 'fill-and-draw' concept using the basal leachate drainage system of landfills as a potential accelerated landfill remediation technique. However, modelling results suggest low contaminant removal efficiency. Including a pause between the fill and the draw cycles improves mass removal., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Tristan Rees-White reports financial support was provided by Engineering and Physical Sciences Research Council. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.]., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Electrical stimulation (EStim), whether used alone or in combination with bone tissue engineering (BTE) approaches, has been shown to promote bone healing. In our previous in vitro studies, mesenchymal stem cells (MSCs) were exposed to EStim and a sustained, long-lasting increase in osteogenic activity was observed. Based on these findings, we hypothesized that pretreating MSC with EStim, in 2D or 3D cultures, before using them to treat large bone defects would improve BTE treatments. Critical size femur defects were created in 120 Sprague-Dawley rats and treated with scaffold granules seeded with MSCs that were pre-exposed or not (control group) to EStim 1 h/day for 7 days in 2D (MSCs alone) or 3D culture (MSCs + scaffolds). Bone healing was assessed at 1, 4, and 8 weeks post-surgery. In all groups, the percentage of new bone increased, while fibrous tissue and CD68+ cell count decreased over time. However, these and other healing features, like mineral density, bending stiffness, the amount of new bone and cartilage, and the gene expression of osteogenic markers, did not significantly differ between groups. Based on these findings, it appears that the bone healing environment could counteract the long-term, pro-osteogenic effects of EStim seen in our in vitro studies. Thus, EStim seems to be more effective when administered directly and continuously at the defect site during bone healing, as indicated by our previous studies.

Chemotherapy-induced peripheral neuropathy (CIPN) is a major unmet medical need with limited treatment options. Despite different mechanisms of action, diverse chemotherapeutics can cause CIPN through a converged pathway─an active axon degeneration program that engages the dual leucine zipper kinase (DLK). DLK is a neuronally enriched kinase upstream in the MAPK-JNK cascade, and while it is dormant under physiological conditions, DLK mediates a core mechanism for neuronal injury response under stress conditions, making it an attractive target for treatment of neuronal injury and neurodegenerative diseases. We have developed potent, selective, brain penetrant DLK inhibitors with excellent PK and activity in mouse models of CIPN. Lead compound IACS-52825 ( 22 ) showed strongly effective reversal of mechanical allodynia in a mouse model of CIPN and was advanced into preclinical development.

Activation of PKG1α is a compelling strategy for the treatment of cardiovascular diseases. As the main effector of cyclic guanosine monophosphate (cGMP), activation of PKG1α induces smooth muscle relaxation in blood vessels, lowers pulmonary blood pressure, prevents platelet aggregation, and protects against cardiac stress. The development of activators has been mostly limited to cGMP mimetics and synthetic peptides. Described herein is the optimization of a piperidine series of small molecules to yield activators that demonstrate in vitro phosphorylation of vasodilator-stimulated phosphoprotein as well as antiproliferative effects in human pulmonary arterial smooth muscle cells. Hydrogen/deuterium exchange mass spectrometry experiments with the small molecule activators revealed a mechanism of action consistent with cGMP-induced activation, and an X-ray co-crystal structure with a construct encompassing the regulatory domains illustrated a binding mode in an allosteric pocket proximal to the low-affinity cyclic nucleotide-binding domain.

A description and the performance of the very small angle neutron scattering diffractometer at the National Institute of Standards and Technology are presented. The measurement range of the instrument extends over three decades of momentum transfer q from 2 × 10 -4 to 0.7 Å -1 . The entire scattering angle range from 8 × 10 -5 to π/6 rad (30°) can be measured simultaneously using three separate detector carriages on rails holding nine 2D detector arrays. Versatile choices of collimation options and neutron wavelength selection allow the q resolution and beam intensity to be optimized for the needs of the experiment. High q resolution is achieved using multiple converging-beam collimation with circular pinholes combined with refractive lenses and prisms. Relaxed vertical resolution with much higher beam intensity can be achieved with narrow slit collimation and a broad wavelength range chosen by truncating the moderator source distribution below 4 Å with a Be crystalline filter and above 8 Å with a supermirror deflector. Polarized beam measurements with full polarization analysis are also provided by a high-performance supermirror polarizer and spin flipper, capable of producing flipping ratios of over 100, along with a high-efficiency 3 He polarization analyzer., (© John Barker et al. 2022.)

This study describes a novel series of UDP- N -acetylglucosamine acyltransferase (LpxA) inhibitors that was identified through affinity-mediated selection from a DNA-encoded compound library. The original hit was a selective inhibitor of Pseudomonas aeruginosa LpxA with no activity against Escherichia coli LpxA. The biochemical potency of the series was optimized through an X-ray crystallography-supported medicinal chemistry program, resulting in compounds with nanomolar activity against P. aeruginosa LpxA (best half-maximal inhibitory concentration (IC 50 ) <5 nM) and cellular activity against P. aeruginosa (best minimal inhibitory concentration (MIC) of 4 μg/mL). Lack of activity against E. coli was maintained (IC 50 > 20 μM and MIC > 128 μg/mL). The mode of action of analogues was confirmed through genetic analyses. As expected, compounds were active against multidrug-resistant isolates. Further optimization of pharmacokinetics is needed before efficacy studies in mouse infection models can be attempted. To our knowledge, this is the first reported LpxA inhibitor series with selective activity against P. aeruginosa .

Milk and milk products are an essential part of global nutrition and the world-wide food industry. Studies of milk components using scattering techniques are well documented in the literature. However, those studies focused on the q scattering wavevector region 10 - 3 < q < 2 Å - 1 . This manuscript presents scattering results in the region 3 × 10 - 5 < q < 2 × 10 - 2 Å - 1 , a region that allows the simultaneous study of fat globules and proteins found in commercial food-grade milk. The small and ultra-small angle neutron scattering (SANS and USANS) measurements show that a model based on the Schulz distribution function using uniform spheres was a reasonable choice to successfully fit the scattering features below q = 0.007 Å - 1 . Contrast measurements using D 2 O on whole milk were carried out to distinguish fat from protein signals. Casein micelles were found to have mean diameters of 96 ± 10 nm with 33% polydispersity. The average scattering length density of the micelles varied from -0.04 × 10 -6 Å -2 in homogenized, pasteurized commercial milk to 2.8 ×10 -6 Å -2 with 50% dilution by D 2 O, with a match point of 43 ± 3%, as seen in previous studies. It was found that the average diameter of fat globules in homogenized whole milk was 0.47 ± 0.04 μm with a polydispersity of 45 ± 5%, and a volume fraction of 0.034 ± 0.002 when the scattering length density is fixed at 0.20 × 10 -6 Å -2 . These USANS measurements provide an important foundation as similar techniques are employed to study cheese varieties and cheese formation.

In situ measurements of geological materials under compression and with hydrostatic fluid pressure are important in understanding their behavior under field conditions, which in turn provides critical information for application-driven research. In particular, understanding the role of nano- to micro-scale porosity in the subsurface liquid and gas flow is critical for the high-fidelity characterization of the transport and more efficient extraction of the associated energy resources. In other applications, where parts are produced by the consolidation of powders by compression, the resulting porosity and crystallite orientation (texture) may affect its in-use characteristics. Small-angle neutron scattering (SANS) and ultra SANS are ideal probes for characterization of these porous structures over the nano to micro length scales. Here we show the design, realization, and performance of a novel neutron scattering sample environment, a specially designed compression cell, which provides compressive stress and hydrostatic pressures with effective stress up to 60 MPa, using the neutron beam to probe the effects of stress vectors parallel to the neutron beam. We demonstrate that the neutron optics is suitable for the experimental objectives and that the system is highly stable to the stress and pressure conditions of the measurements.

In order to reduce pain caused by the affected hip joint, unilateral hip osteoarthritis patients (HOAP) adopt characteristic gait patterns. However, it is unknown if the knee and hip joint loading in the non-affected (limb non-affected ) and the affected (limb affected ) limb differ from healthy controls (HC) and which gait parameters correlate with potential abnormal joint loading. Instrumented 3D-gait analysis was performed on 18 HOAP and 18 sex, age, and height matched HC. The limb non-affected showed greater first and second peak external hip adduction moments (first HAM: +15%, p = 0.014; second HAM: +15%, p = 0.021, respectively), than seen in HC. In contrast, the second peak external knee adduction moment (KAM) in the limb affected is reduced by about 23% and 30% compared to the limb non-affected and HC, respectively. Furthermore, our patients showed characteristic gait compensation strategies including reduced peak vertical forces (pvF), a greater foot progression angle (FPA), and reduced knee range of motion (ROM) in the limb affected . The limb affected was 5.6 ± 3.8 mm shorter than the limb non-affected . Results of stepwise regression analyses showed that increased first pvF explain 16% of first HAM alterations, whereas knee ROM and FPA explain 39% of second KAM alterations. We therefore expect an increased rate of progression of OA in the hip joint of the limb non-affected and suggest that the shift in the medial-to-lateral knee joint load distribution may impact the rate of progression of OA in the limb affected . The level of evidence is III. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1764-1773, 2017., (© 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

The emergence and spread of multidrug-resistant (MDR) Gram negative bacteria presents a serious threat for public health. Novel antimicrobials that could overcome the resistance problems are urgently needed. UDP-3-O-(R-3-hydroxymyristol)-N-acetylglucosamine deacetylase (LpxC) is a cytosolic zinc-based deacetylase that catalyzes the first committed step in the biosynthesis of lipid A, which is essential for the survival of Gram-negative bacteria. Our efforts toward the discovery of novel LpxC inhibitors are presented herein., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The objective of this study was to test if patients with unilateral hip osteoarthritis (OA) show greater muscle activity asymmetry between their affected and non-affected limbs than healthy controls between their left and right limbs. Seventeen patients with unilateral hip OA (7 females, 10 males) and 17 age-matched healthy controls (7 females, 10 males) participated in this study. Both groups performed instrumented gait analysis at comparable speeds. Muscle activity was recorded simultaneously for the tibialis anterior (TA), gastrocnemius medialis (GM), vastus lateralis (VL), semitendinosus (ST), tensor fasciae latae (TFL), and gluteus medius (GLM) muscles. In hip OA patients, EMG data showed greater activity of the TA muscle in the non-affected limb, and greater TFL muscle activity in the affected limb. Compared to healthy controls, greater asymmetries between paired limbs were observed for the TA and GM muscles. Finally, the TFL muscle of the affected limb contributed more to the total limb muscle activity than did the non-affected limb. The observed alterations in TA and GM muscle activity in hip OA patients may be due to the greater peak braking and peak vertical forces measured in the non-affected limb. Contrary to this, greater TLF muscle activity of the affected limb indicates the demands put on stabilizing the hip during stance phase. Further studies are necessary to test whether leg length discrepancy affects muscle activation alterations between the affected and non-affected limb in unilateral hip OA patients., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The ATPase subunit of DNA gyrase B is an attractive antibacterial target due to high conservation across bacteria and the essential role it plays in DNA replication. A novel class of pyrazolopyridone inhibitors was discovered by optimizing a fragment screening hit scaffold using structure guided design. These inhibitors show potent Gram-positive antibacterial activity and low resistance incidence against clinically important pathogens.

Inhibitors of the ATPase function of bacterial DNA gyrase, located in the GyrB subunit and its related ParE subunit in topoisomerase IV, have demonstrated antibacterial activity. In this study we describe an NMR fragment-based screening effort targeting Staphylococcus aureus GyrB that identified several attractive and novel starting points with good ligand efficiency. Fragment hits were further characterized using NMR binding studies against full-length S. aureus GyrB and Escherichia coli ParE. X-ray co-crystal structures of select fragment hits confirmed binding and suggested a path for medicinal chemistry optimization. The identification, characterization, and elaboration of one of these fragment series to a 0.265 μM inhibitor is described herein., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

If you are subject to an investigation by a professional regulator, you need to be informed about the process and act quickly. This article outlines the process and offers some advice on how best to navigate it.

Introduction: Cancellous bone is frequently used for filling bone defects in a clinical setting. It provides favourable conditions for regenerative cells such as MSC and early EPC. The combination of MSC and EPC results in superior bone healing in experimental bone healing models., Materials and Methods: We investigated the influence of osteogenic culture conditions on the endothelial properties of early EPC and the osteogenic properties of MSC when cocultured on cancellous bone. Additionally, cell adhesion, metabolic activity, and differentiation were assessed 2, 6, and 10 days after seeding., Results: The number of adhering EPC and MSC decreased over time; however the cells remained metabolically active over the 10-day measurement period. In spite of a decline of lineage specific markers, cells maintained their differentiation to a reduced level. Osteogenic stimulation of EPC caused a decline but not abolishment of endothelial characteristics and did not induce osteogenic gene expression. Osteogenic stimulation of MSC significantly increased their metabolic activity whereas collagen-1α and alkaline phosphatase gene expressions declined. When cocultured with EPC, MSC's collagen-1α gene expression increased significantly., Conclusion: EPC and MSC can be cocultured in vitro on cancellous bone under osteogenic conditions, and coculturing EPC with MSC stabilizes the latter's collagen-1α gene expression.

Background: Clara cell protein 16 (CC16) has recently gained acceptance as a blood biomarker for detecting direct and indirect lung injury. Although the early elevation of CC16 serum levels has been shown to correlate with pulmonary damage in patients with multiple injuries, the subsequent time course of CC16 serum levels has not been investigated in these patients., Methods: Fifty-eight patients with multiple injuries, 32 with severe thoracic injury, and 12 healthy volunteers were enrolled in this study. CC16 serum levels were measured at the time they were admitted to the trauma ward "time 0" and subsequently until day 14 using the enzyme-linked immunosorbent assay technique. The correlation between CC16 serum levels and severe lung injury, onset of nosocomial pneumonia, acute respiratory distress syndrome or acute lung injury, and organ failure was measured. In addition, areas under the receiver operating characteristic curve were calculated (p < 0.05 = significant)., Results: In patients with lung injury, initial "time 0" median CC16 values were significantly elevated (11.2 ng/mL) compared with patients without severe thoracic injury (6.9 ng/mL) and controls (6.3 ng/mL). The observed elevation in serum CC16 declined to control values within 12 to 24 hours after trauma unless patients secondarily developed pneumonia. In the latter patients, median CC16 serum levels were significantly elevated (14.5 ng/mL) at the onset of pneumonia compared with their levels (7.3 ng/mL) 1 day before. In contrast, no secondary elevation in CC16 serum levels was observed in patients without severe lung injury within the same 24-hour period. The area under the receiver operating characteristic curve for serum CC16 and pneumonia was 0.79 (0.62-0.97; p = 0.0011)., Conclusion: Our results confirm the previously described association between initial elevation in CC16 serum levels and severe thoracic injury in patients with multiple injuries. In addition, we found that the initial elevation in CC16 serum levels declines to control values within the first day after trauma and that a secondary elevation indicates respiratory complications., Level of Evidence: Diagnostic study, level II.

Background: Pulmonary complications are common in multiple trauma patients with chest injury. Factors predisposing these critically ill patients to respiratory organ failure are not fully understood., Methods: Univariate and multivariate logistic regression analyses were used to assess the prognostic value of clinical and laboratory variables (2002-2008; n = 30,616) from the Trauma Registry of the German Trauma Society (DGU). Data from patients admitted to the ICU with lung contusion/lacerations, an Injury Severity Score ≥ 16 and age ≥ 18 were included in the study. Severe pulmonary organ failure was defined as PaO(2)/FiO(2)<200 for ≥ 3 days and based on the odds ratios (ORs) a simplified Lung Organ Failure Score (LOFS) was developed using integer values., Results: 21.3% (1254) of the 5892 patients analysed developed severe pulmonary organ failure. We identified seven independent predictors with significant correlation: age, gender, head injury, fluid therapy, injury severity, degree of chest trauma and surgical interventions. The highest ORs were observed in cases of Abbreviated Injury Scale (AIS)(Thorax) = 5 (1.58), surgical intervention (1.71) and multiple surgeries (2.41). We found that patients with simplified score values ≥ 21 points were at a maximum risk (>30%) for developing severe pulmonary complications., Conclusion: This scoring method could help trauma surgeons determine which multiple trauma patients are at risk for pulmonary complications after trauma. Efficacy analyses of prophylactic PEEP ventilation or rotational bed therapy in subgroups with comparable risks for respiratory complication could be based on the LOFS., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Endothelial progenitor cells (EPCs) and polymorphonucleated leukocytes (PMNLs) migrate to and accumulate at the site of tissue injury where they express complementary sets of surface receptors (CD11b/CD18, CD54), suggesting a possible cellular interaction. Trauma-activated PMNLs release inflammatory mediators and reactive oxygen species (ROS) produced by the NADPH oxidase, which may negatively impact EPCs. To characterize the interactions between PMNLs and EPCs, we identified common surface receptors and measured the role played by NADPH oxidase and neutrophil elastase. Polymorphonucleated leukocytes were obtained from either healthy volunteers or multiple-trauma patients. After stimulation with either n-formyl-l-methionyl-l-leucyl-l-phenylalanine or phorbol 12-myristate 13-acetate, the PMNLs were incubated with DiL-prestained EPCs in a ratio of 20:1 for 3 h. Early EPCs were isolated from buffy coat. Endothelial progenitor cell killing was measured by flow cytometry, and necrotic EPCs were identified by measuring the uptake of 7-aminoactinomycin. We found that blocking CD11b, CD18, or CD54 on the EPC surface with monoclonal antibodies or blocking the intracellular production of ROS by neutralizing neutrophil's NADPH oxidase with a diphenyliodonium chloride pretreatment protected EPCs, enhancing its survival, whereas inhibiting neutrophil elastase had no effect on survival. Furthermore, we observed that native PMNLs obtained from multiple-trauma patients damaged EPCs, whereas native PMNLs from healthy volunteers did not. Our results demonstrate that EPCs and PMNLs do interact via complementary receptors and that this interaction results in PMNL-derived ROS-induced EPC damage. The effect of neutrophil-derived elastase was found to be negligible. These findings suggest that EPC damage by activated PMNLs may contribute to impaired wound healing observed after severe trauma.

The linking together of two fragment compounds that bind to distinct protein sub-sites can lead to a superadditivity of binding affinities, in which the binding free energy of the linked fragments exceeds the simple sum of the binding energies of individual fragments (linking coefficient E<1). However, a review of the literature shows that such events are relatively rare and, in the majority of the cases, linking coefficients are far from optimal being much greater than 1. It is critical to design a linker that does not disturb the original binding poses of each fragment in order to achieve successful linking. However, such an ideal linker is often difficult to design and even more difficult to actually synthesize. We suggest that the chance of achieving successful fragment linking can be significantly improved by choosing a fragment pair that consists of one fragment that binds by strong H-bonds (or non-classical equivalents) and a second fragment that is more tolerant of changes in binding mode (hydrophobic or vdW binders). We also propose that the fragment molecular orbital (FMO) calculations can be used to analyse the nature of the binding interactions of the fragment hits for the selection of fragments for evolution, merging and linking in order to optimize the chance of success., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Research in the geographies of care has identified the central role of mothers in caring for children, although much less explored are the experiences of men who also participate in care. Drawing upon research conducted in the UK with children and their families, this paper contributes to existing debates in the geographies of care by exploring a relatively new space of caring, namely the escort of primary school children to and from school and other settings. The paper explores mothers' and fathers' involvement in escorting children, the extent and nature of participation and also how distinct gendered forms of caring practices are established. In doing so, the paper also considers the importance of place and local cultures of parenting which inform these gendered carescapes., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Background: Impaired wound healing due to local injury, infection, or systemic diseases, such as diabetes, is a major clinical problem. Recent studies have shown that endothelial progenitor cells (EPC) isolated from peripheral blood, bone marrow, as well as the spleen accumulate in granulation tissue at the site of neovascularization, causing secretion of growth factors and cytokines and thus accelerating wound healing., Materials and Methods: In the present study, we transplanted systemic EPC and then measured epithelialization and neovascularization in the hairless mouse ear wound model., Results: Systemic EPC transplantation significantly accelerated epithelialization and neovascularization compared with control wounds receiving phosphate-buffered saline without calcium and magnesium (PBS). The EPC group had significantly higher vascular density than did the PBS-treated group as determined by immunohistochemistry for CD31 and CD90. Fluorescence microscopy revealed accumulation "homing" of the transplanted EPC at the sites of neovascularization in the granulation tissue throughout healing. Furthermore, transplantation of EPC also increased the expression of the angiogenic cytokine stromal cell-derived factor 1α (SDF1α)., Conclusions: This appears to be the first demonstration of EPC recruitment to the site of wound neovascularization throughout the healing process. These findings demonstrate that transplanting systemic EPC into "normal" healing wounds promotes epithelialization and neovascularization and thus could be an useful method for accelerating wound healing., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The mammalian target of rapamycin (mTOR) is a Ser/Thr protein kinase and a major controller of cell growth. In cells, mTOR forms two distinct multiprotein complexes, mTORC1 and mTORC2. The mTORC1 complex can phosphorylate 4EBP1 and S6K1, two key regulators of translation initiation, whereas mTORC2 phosphorylates AKT1, an event required for AKT1 activation. Here, we expressed and purified human mTORC1 and mTORC2 from HEK-293 cells using FLAG-M2 affinity chromatography. Western blotting analysis using phospho-specific antibodies indicated that recombinant mTORC1 and mTORC2 exhibit distinct substrate preferences in vitro, consistent with their roles in cells. To improve our understanding of the enzymatic properties of mTOR alone and mTOR in its complex form, steady-state kinetic profiles of truncated mTOR containing the kinase domain (residues 1360-2549) and mTORC1 were determined. The results revealed that mTORC1 is catalytically less active than truncated mTOR, as evidenced by 4.7- and 3.1-fold decreases in catalytic efficiency, k(cat)/K(m), for ATP and 4EBP1, respectively. We also found that truncated mTOR undergoes autophosphorylation through an intramolecular mechanism. Mass spectrometric analysis identified two novel mTOR autophosphorylation sites, Ser2454 and either Thr2473 or Thr2474, in addition to the previously reported Ser2481 site. Truncated mTOR and mTORC1 were completely inhibited by ATP competitive inhibitors PI103 and BEZ235 and partially inhibited by rapamycin/FKBP12 in a noncompetitive fashion toward ATP. All inhibitors tested exhibited similar inhibitory potencies between mTORC1 and truncated mTOR containing the kinase domain. Our studies presented here provide the first detailed kinetic studies of a recombinant mTOR complex.

A novel series of 2-aminobenzimidazole inhibitors of BACE1 has been discovered using fragment-based drug discovery (FBDD) techniques. The rapid optimization of these inhibitors using structure-guided medicinal chemistry is discussed., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Ischemia and reperfusion of organs is an unavoidable consequence of transplantation. Inflammatory events associated with reperfusion injury are in part attributed to excessive complement activation. Systemic administration of complement inhibitors reduces reperfusion injury but leaves patients vulnerable to infection. Here, we report a novel therapeutic strategy that decorates cells with an anti-complement peptide. An analog of the C3 convertase inhibitor Compstatin (C) was synthesized with a hexahistidine (His(6)) tag to create C-His(6). To decorate cell membranes with C-His(6), fusogenic lipid vesicles (FLVs) were used to incorporate lipids with nickel (Ni(2+)) tethers into cell membranes, and these could then couple with C-His(6). Ni(2+) tether levels to display C-His(6) were modulated by changing FLV formulation, FLV incubation time and FLV levels. SKOV-3 cells decorated with C-His(6) effectively reduced complement deposition in a classical complement activation assay. We conclude that our therapeutic approach appears promising for local ex vivo treatment of transplanted organs to reduce complement-mediated reperfusion injury.

Serine racemase is responsible for the synthesis of D-serine, an endogenous co-agonist for N-methyl-D-aspartate receptor-type glutamate receptors (NMDARs). This pyridoxal 5'-phosphate-dependent enzyme is involved both in the reversible conversion of L- to D-serine and serine catabolism by alpha,beta-elimination of water, thereby regulating D-serine levels. Because D-serine affects NMDAR signaling throughout the brain, serine racemase is a promising target for the treatment of disorders related to NMDAR dysfunction. To provide a molecular basis for rational drug design the x-ray crystal structures of human and rat serine racemase were determined at 1.5- and 2.1-A resolution, respectively, and in the presence and absence of the orthosteric inhibitor malonate. The structures revealed a fold typical of beta-family pyridoxal 5'-phosphate enzymes, with both a large domain and a flexible small domain associated into a symmetric dimer, and indicated a ligand-induced rearrangement of the small domain that organizes the active site for specific turnover of the substrate.

Deeply weathered crystalline rock aquifer systems comprising unconsolidated saprolite and underlying fractured bedrock (saprock) underlie 40% of sub-Saharan Africa. The vulnerability of this aquifer system to contamination, particularly in rapidly urbanizing areas, remains poorly understood. In order to assess solute and viral transport in saprolite derived from Precambrian gneiss, forced-gradient tracer experiments using chloride and Escherichia coli phage PhiX174 were conducted in southeastern Uganda. The bacteriophage tracer was largely unrecovered; adsorption to the weathered crystalline rock matrix is inferred and enabled by the low pH (5.7) of site ground water and the bacteriophage's relatively high isoelectric point (pI = 6.6). Detection of the applied PhiX174 phage in the pumping well discharge at early times during the experiment traces showed, however, that average ground water flow velocities exceed that of the inert solute tracer, chloride. This latter finding is consistent with observations in other hydrogeological environments where statistically extreme sets of microscopic flow velocities are considered to transport low numbers of fecal pathogens and their proxies along a selected range of linked ground water pathways. Application of a radial advection-dispersion model with an exponentially decaying source term to the recovered chloride tracer estimates a dispersivity (alpha) of 0.8 +/- 0.1 m over a distance of 4.15 m. Specific yield (S(y)) is estimated to be 0.02 from volume balance calculations based on tracer experiments. As single-site observations, our estimates of saprolite S(y) and alpha are tentative but provide a starting point for assessing the vulnerability of saprolite aquifers in sub-Saharan Africa to contamination and estimating quantitatively the impact of climate and abstraction on ground water storage.

The Mapkap kinases 2 and 3 (MK2 and MK3) have been implicated in intracellular signaling pathways leading to the production of the pro-inflammatory cytokine tumor necrosis factor alpha. MK2 has been pursued by the biopharmaceutical industry for many years for the development of a small molecule anti-inflammatory treatment and drug-like inhibitors have been described. The development of some of these compounds, however, has been slowed by the absence of a high-resolution crystal structure of MK2. Herein we present a high-resolution (1.9 A) crystal structure of the highly homologous MK3 in complex with a pharmaceutical lead compound. While all of the canonical features of Ser/Thr kinases in general and MK2 in particular are recapitulated in MK3, the detailed analysis of the binding interaction of the drug-like ligand within the adenine binding pocket allows relevant conclusions to be drawn for the further design of potent and selective drug candidates.