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Frontal affinity chromatography is a powerful, underappreciated technique for the qualitative (screening) and quantitative ( K d determination) evaluation of biological interactions. Its development has been previously hampered by its sample consumption, limited throughput, and lack of dedicated instrumentation especially at a miniaturized scale. This work describes two original experimental devices allowing nano-frontal affinity chromatography titrations (nano-FAC) to be automatically implemented in the time-saving staircase mode. The first nano-FAC system utilizes a capillary electrophoresis device (7100 CE Agilent system) in the pressurization mode with in situ UV detection. The second nano-FAC experimental setup implements a nano-LC device (Ultimate 3000 Thermo) modified with a 10-port valve equipped with two superloops (loop volume, 5 μL) operating alternatively and automatically in a single run. The benefits and drawbacks of each approach are exemplified using two model protein-ligand interactions (concanavalin A-mannose and concanavalin A-glucose). The two methods result in concordant dissociation constants ( K d ) and number of active site ( B act ) values, obtained in a fully automated manner, with low sample consumption and good throughput.

Here we describe a new HPLC-MS/MS method using a mixed mode stationary phase and a binary gradient of elution for the rapid separation and quantification of AAs in human plasma without derivatization or ion pairing reagent addition. The sample preparation procedure consists in a single dilution step after protein precipitation with sulfosalicylic acid. The proposed method allows for the unambiguous identification and analysis of 52 AAs and related compounds including the separation of isomers and isobars in an 18 min chromatographic run including the conditioning and the equilibration times. AAs were detected by selective reaction monitoring. Internal calibration was used for the quantification of 37 AAs, including 25 using the corresponding isotopically labeled internal standards. External calibration (no internal standard) was used for five additional analytes. Qualitative detection was achieved for the remaining compounds. Validation studies evaluated accuracy, linearity, within- and between-run precision, lower limits of detection and quantification for 37 amino acids present in commonly used quality control samples. For within-run precision CVs averaged 3.8 % (n = 30) for all compounds. For between-run precision, CVs averaged 8.6 % for all compounds (n = 20). Correlation with the common standard ion-exchange chromatography with post-column derivatization method was also performed for 32 plasma samples. While the proposed method is at least 50 times more sensitive, the data showed good correlation with slopes equal or higher than 0.9 and correlation coefficients mostly higher than 0.90. The method was successfully applied for analysis of plasma samples for detection of inherited disorders of amino acid metabolism., Competing Interests: Declaration of Competing Interest The authors 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 © 2020 Elsevier B.V. All rights reserved.)

Dendrogenin A (DDA) was recently identified as a mammalian cholesterol metabolite that displays tumor suppressor and neurostimulating properties at low doses. In breast tumors, DDA levels were found to be decreased compared to normal tissues, evidencing a metabolic deregulation of DDA production in cancers. DDA is an amino-oxysterol that contains three protonatable nitrogen atoms. This makes it physico-chemically different from other oxysterols and it therefore requires specific analytical methods We have previously used a two-step method for the quantification of DDA in biological samples: 1) DDA purification from a Bligh and Dyer extract by RP-HPLC using a 250×4.6mm column, followed by 2) nano-electrospray ionization mass spectrometry (MS) fragmentation to analyze the HPLC fraction of interest. We report here the development a liquid chromatography tandem mass spectrometry method for the analysis of DDA and its analogues. This new method is fast (10min), resolving (peak width <4s) and has a weak carryover (<0.01%). We show that this technique efficiently separates DDA from its C17 isomer and other steroidal alkaloids from the same family establishing a proof of concept for the analysis of this family of amino-oxysterols., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Radiation therapy plays an essential part in modern treatment regimes of musculoskeletal tumors. Nevertheless damage to the surrounding tissue does occur inevitably. Postradiogenic changes of bone are associated with decreased stability and an increased fracture rate. The orthopedic surgeon therefore faces a challenging situation with altered bone metabolism, changes in perfusion and soft tissue problems., Patients/material and Methods: We present 3 cases of radiation induced fractures during the treatment of soft tissue tumors, all of which received radiation doses of > 58 Gy. All fractures occurred over 1 year after the exposure to radiation in otherwise uneventful follow ups., Results: Postoperative follow up showed fracture healing or in the case of the arthroplasty, osseous integration without further complications., Conclusions: Radiation doses of ≥ 58 Gy are a major risk factor for pathological fractures in long bones. Regardless of their low incidence, fracture rates between 1,2 and 6,4 % prove their importance. Local tumor control has therefore to be weighed against the resulting decrease in bone quality and stability. Treatment options should always take into consideration the increased risk for complications such as infection, pseudarthroses and wound healing disorders. Our results show that substitution of vitamin D and calcium as well as the the use of reamed intramedullary implants benefits the outcome.

This study assessed the Anti-Stigma Project workshop, a contact/education intervention developed by On Our Own of Maryland, Inc. and the Maryland Mental Hygiene Administration. Two separate randomized controlled trials administered pre- and post-test questionnaire assessments. One included people with mental illness (N = 127) and a second included mental health providers (N = 131). Post-intervention, people with mental illness were more aware of stigma, had lower levels of prejudice, and increased belief in recovery. Providers were more aware of stigma, had lower levels of prejudice, and increased concurrence in self-determination of people with mental illness. Increasing providers' stigma awareness and recognition can promote higher quality service delivery. Increasing stigma awareness and recognition for people with mental illness can foster confidence in overcoming psychiatric disabilities. Using a participatory action research team, our protocol included extant and newly developed stigma change tools. Organizations seeking to conduct effective evaluation studies should consider collaborative processes including the expertise of affected constituents.

Parathyroid hormone (PTH) is only one measurable index of skeletal health, and we reasoned that a histomorphometric analysis of iliac crest biopsies would be another and even more direct approach to assess bone health and address the required minimum 25-Hydroxyvitamin D [25(OH)D] level. A cohort from the northern European population with its known high prevalence of vitamin D deficiency therefore would be ideal to answer the latter question. We examined 675 iliac crest biopsies from male and female individuals, excluding all patients who showed any signs of secondary bone diseases at autopsy. Structural histomorphometric parameters, including osteoid indices, were quantified using the Osteomeasure System according to ASBMR standards, and serum 25(OH)D levels were measured for all patients. Statistical analysis was performed by Student's t test. The histologic results demonstrate an unexpected high prevalence of mineralization defects, that is, a pathologic increase in osteoid. Indeed, 36.15% of the analyzed patients presented with an osteoid surface per bone surface (OS/BS) of more than 20%. Based on the most conservative threshold that defines osteomalacia at the histomorphometric level with a pathologic increase in osteoid volume per bone volume (OV/BV) greater than 2% manifest mineralization defects were present in 25.63% of the patients. The latter were found independent of bone volume per trabecular volume (BV/TV) throughout all ages and affected both sexes equally. While we could not establish a minimum 25(OH)D level that was inevitably associated with mineralization defects, we did not find pathologic accumulation of osteoid in any patient with circulating 25(OH)D above 75 nmol/L. Our data demonstrate that pathologic mineralization defects of bone occur in patients with a serum 25(OH)D below 75 nmol/L and strongly argue that in conjunction with a sufficient calcium intake, the dose of vitamin D supplementation should ensure that circulating levels of 25(OH)D reach this minimum threshold (75 nmol/L or 30 ng/mL) to maintain skeletal health., (Copyright 2010 American Society for Bone and Mineral Research.)

Autoinhibition is a recurring mode of protein kinase regulation and can be based on diverse molecular mechanisms. Here, we show by crystal structure analysis, nuclear magnetic resonance (NMR)-based nucleotide affinity studies and rational mutagenesis that nonphosphorylated mitogen-activated protein (MAP) kinases interacting kinase (Mnk) 1 is autoinhibited by conversion of the activation segment into an autoinhibitory module. In a Mnk1 crystal structure, the activation segment is repositioned via a Mnk-specific sequence insertion at the N-terminal lobe with the following consequences: (i) the peptide substrate binding site is deconstructed, (ii) the interlobal cleft is narrowed, (iii) an essential Lys-Glu pair is disrupted and (iv) the magnesium-binding loop is locked into an ATP-competitive conformation. Consistently, deletion of the Mnk-specific insertion or removal of a conserved phenylalanine side chain, which induces a blockade of the ATP pocket, increase the ATP affinity of Mnk1. Structural rearrangements required for the activation of Mnks are apparent from the cocrystal structure of a Mnk2 D228G -staurosporine complex and can be modeled on the basis of crystal packing interactions. Our data suggest a novel regulatory mechanism specific for the Mnk subfamily.

Human mitogen-activated protein kinases (MAPK)-interacting kinases 1 and 2 (Mnk1 and Mnk2) target the translational machinery by phosphorylation of the eukaryotic initiation factor 4E (eIF4E). Here, we present the 2.1 A crystal structure of a nonphosphorylated Mnk2 fragment that encompasses the kinase domain. The results show Mnk-specific features such as a zinc binding motif and an atypical open conformation of the activation segment. In addition, the ATP binding pocket contains an Asp-Phe-Asp (DFD) in place of the canonical magnesium binding Asp-Phe-Gly (DFG) motif. The phenylalanine of this motif sticks into the ATP binding pocket and blocks ATP binding as observed with inhibitor bound and, thus, inactive p38 kinase. Replacement of the DFD by the canonical DFG motif affects the conformation of Mnk2, but not ATP binding and kinase activity. The results suggest that the ATP binding pocket and the activation segment of Mnk2 require conformational switches to provide kinase activity.

We describe enzymological and structural analyses of the interaction between the family 18 chitinase ChiB from Serratia marcescens and the designed inhibitor N,N'-diacetylchitobionoxime-N-phenylcarbamate (HM508). HM508 acts as a competitive inhibitor of this enzyme with a K(i) in the 50 microM range. Active site mutants of ChiB show K(i) values ranging from 1 to 200 microM, providing insight into some of the interactions that determine inhibitor affinity. Interestingly, the wild type enzyme slowly degrades HM508, but the inhibitor is essentially stable in the presence of the moderately active D142N mutant of ChiB. The crystal structure of the D142N-HM508 complex revealed that the two sugar moieties bind to the -2 and -1 subsites, whereas the phenyl group interacts with aromatic side chains that line the +1 and +2 subsites. Enzymatic degradation of HM508, as well as a Trp --> Ala mutation in the +2 subsite of ChiB, led to reduced affinity for the inhibitor, showing that interactions between the phenyl group and the enzyme contribute to binding. Interestingly, a complex of enzymatically degraded HM508 with the wild type enzyme showed a chitobiono-delta-lactone bound in the -2 and -1 subsites, despite the fact that the equilibrium between the lactone and the hydroxy acid forms in solution lies far toward the latter. This shows that the active site preferentially binds the (4)E conformation of the -1 sugar, which resembles the proposed transition state of the reaction.