Bactericidal activity of extended 9-glycyl-amido-minocyclines

The need for self-protecting polymer or alloy implants resistant to a broad spectrum of bacterial challenges led us to investigate covalent bonding of minocycline (MIN), a tetracycline derivative, to polystyrene beads and to titanium alloy foils by oligoethylene glycol spacers. 9-Hydrazino-acetyl-amido-MIN, and simpler glycylcycline derivatives, retained minimum inhibitory concentration (MIC) against Staphylococcus aureus comparable to MIN. However, PEG-glycyl-amido-MIN showed very low activity. Hence, we coupled 9-hydrazino-acetyl-amido-MIN to the aldehyde termini of oligoethylene glycol spacers bonded to polystyrene and titanium alloy surfaces to form acid-releasable hydrazone linkages. 9-Hydrazino-acetyl-amido-MIN was released from the monolayers more rapidly at pH 5.0 than at pH 7.4

Molecular rulers for measuring RNA structure: sites of crosslinking in chlorambucilyl-phenylalanyl-tRNA^(Phe) (yeast) and chlorambucilyl-pentadecaprolyl-phenylalanyl-tRNA^(Phe) (yeast) intramolecularly crosslinked in aqueous solution

Intramolecular crosslinking of yeast phenylalanine tRNA in aqueous solution with rigid, variable-length crosslinking reagents, which we call "molecular rulers," has given results in reasonable agreement with the crystal structure. Chlorambucilyl-[^(3)H]phenylalanyl-tRNA^(Phe) crosslinked intramolecularly at G-71 and A-73, whereas chlorambucilyl-pentadecaprolyl-[^(3)H]phenylalanyl-tRNA^(Phe) crosslinked at G-20 and Y-37. The pentadecaprolyl reagent was predicted to be 62 Å long, including chlorambucil and phenylalanine; the sites that it reached are 60 Å distant from the 3' OH (in the case of G-20) or 80 Å distant (in the case of Y-37) in the crystal structure of tRNA^(Phe). The close agreement between the length of the reagent and the distance of G-20 from the 3' OH in the crystal structure illustrates the rigidity of the tRNA^(Phe) molecule in the dihydrouridine loop region at the corner of the molecule. The apparent ability of the 62-Å-long reagent to crosslink to a site, Y-37, that is 80 Å distant from the 3' OH in the crystal structure appears to illustrate the flexibility of both the 3' A-C-C-A terminus and the anticodon stem and loop, with respect to the tRNA molecule. These observations demonstrate the utility of oligoproline-based crosslinking reagents as rigid, variable-length molecular rulers for biological macromolecules in solution

Limited tolerance towards folded elements during secretion of the autotransporter Hbp

Many virulence factors secreted by pathogenic Gram-negative bacteria belong to the autotransporter (AT) family. ATs consist of a passenger domain, which is the actual secreted moiety, and a β-domain that facilitates the transfer of the passenger domain across the outer membrane. Here, we analysed folding and translocation of the AT passenger, using Escherichia coli haemoglobin protease (Hbp) as a model protein. Dual cysteine mutagenesis, instigated by the unique crystal structure of the Hbp passenger, resulted in intramolecular disulphide bond formation dependent on the periplasmic enzyme DsbA. A small loop tied off by a disulphide bond did not interfere with secretion of Hbp. In contrast, a bond between different domains of the Hbp passenger completely blocked secretion resulting in degradation by the periplasmic protease DegP. In the absence of DegP, a translocation intermediate accumulated in the outer membrane. A similar jammed intermediate was formed upon insertion of a calmodulin folding moiety into Hbp. The data suggest that Hbp can fold in the periplasm but must retain a certain degree of flexibility and/or modest width to allow translocation across the outer membrane

Tweeting with Authority: Identifying Influential Participants in Agriculture-Related Water Quality Twitter Conversations

In August 2014, Toledo, Ohio, experienced a dangerous algae bloom that led to a citywide water ban. News media coverage of the incident was widespread, as was social media conversation. Opinion leadership has been linked to news media agenda setting, and social media users are capable of generating considerable social influence within the sphere of their social networks. To better understand the dynamics of the conversation—especially as it pertained to agriculture—both during and after the water ban, the researchers used a social media analysis platform to identify high-influence Twitter users who participated in water-quality discussions about the Toledo water ban. Narrowing the search to agriculture-related discussion, the researchers revealed a dearth of agriculture-related content and also identified three categories of Twitter users in the conversation, including news sources, activists, and agriculture advocates. The researchers also found that Ohio users in post-ban discussions tended to be more influential than those who participated during the water ban. Identifying these users allows practitioners to monitor influential accounts for emerging issues and to engage with authoritative users in their geographic regions. The researchers also recommend that agriculture advocates exercise restraint in publicly speaking out about the industry’s involvement in environmental issues

Where I Come From: Exploring Regional Differences in California Consumers’ Attitudes and Beliefs About Fluid Milk

Guided by the theory of reasoned action and social cognitive theory, this study was conducted to better understand how decisions to purchase organic or conventional milk are influenced by norms and attitudes established via human interactions and how those norms and attitudes vary by geographic region. An online Qualtrics survey was used to gain insight into behaviors, attitudes, and interactions of 308 milk consumers in various geographical regions of [State]. The findings indicate that urban, suburban, and rural consumers differ in some ways in their interactions related to milk information, as well as their perceptions of organic versus conventional milk. Based on the findings of the study, differences in personal networks and exposure to certain messaging in varying regions might perpetuate a more positive, confident, and informed view of organic or conventional milk products in some areas more so than others. The researchers recommend using the results of the study to tailor messages to the specific information needs of consumers in urban, suburban, and rural regions of [State]

Two-pronged attack: dual inhibition of Plasmodium falciparum M1 and M17 metalloaminopeptidases by a novel series of hydroxamic acid-based inhibitors

Plasmodium parasites, the causative agents of malaria, have developed resistance to most of our current antimalarial therapies, including artemisinin combination therapies which are widely described as our last line of defense. Antimalarial agents with a novel mode of action are urgently required. Two Plasmodium falciparum aminopeptidases, PfA-M1 and PfA-M17, play crucial roles in the erythrocytic stage of infection and have been validated as potential antimalarial targets. Using compound-bound crystal structures of both enzymes, we have used a structure-guided approach to develop a novel series of inhibitors capable of potent inhibition of both PfA-M1 and PfA-M17 activity and parasite growth in culture. Herein we describe the design, synthesis, and evaluation of a series of hydroxamic acid-based inhibitors and demonstrate the compounds to be exciting new leads for the development of novel antimalarial therapeutics

Disordered Structural Ensembles of Vasopressin and Oxytocin and Their Mutants

Vasopressin and oxytocin are intrinsically disordered cyclic nonapeptides belonging to a family of neurohypophysial hormones. Although unique in their functions, these peptides differ only by two residues and both feature a tocin ring formed by the disulfide bridge between first and sixth cysteine residues. This sequence and structural similarity are experimentally linked to oxytocin agonism at vasopressin receptors and vasopressin antagonism at oxytocin receptors. Yet single- or double-residue mutations in both peptides have been shown to have drastic impacts on their activities at either receptor, and possibly the ability to bind to their neurophysin carrier protein. In this study we perform molecular dynamics simulations of the unbound native and mutant sequences of the oxytocin and vasopressin hormones to characterize their structural ensembles. We classify the subpopulations of these structural ensembles on the basis of the distributions of radius of gyration and secondary structure and hydrogen-bonding features of the canonical tocin ring and disordered tail region. We then relate the structural changes observed in the unbound form of the different hormone sequences to experimental information about peptide receptor binding, and more indirectly, carrier protein binding affinity, receptor activity, and protease degradation. This study supports the hypothesis that the structural characteristics of the unbound form of an IDP can be used to predict structural or functional preferences of its functional bound form

Targeting the hedgehog transcription factors GLI1 and GLI2 restores sensitivity to vemurafenib-resistant human melanoma cells

BRAF inhibitor (BRAFi) therapy for melanoma patients harboring the V600E mutation is initially highly effective, but almost all patients relapse within a few months. Understanding the molecular mechanisms underpinning BRAFi-based therapy is therefore an important issue. Here we identified a previously unsuspected mechanism of BRAFi resistance driven by elevated Hedgehog (Hh) pathway activation that is observed in a cohort of melanoma patients after vemurafenib treatment. Specifically, we demonstrate that melanoma cell lines, with acquired in vitro-induced vemurafenib resistance, show increased levels of glioma-associated oncogene homolog 1 and 2 (GLI1/GLI2) compared with naive cells. We also observed these findings in clinical melanoma specimens. Moreover, the increased expression of the transcription factors GLI1/GLI2 was independent of canonical Hh signaling and was instead correlated with the noncanonical Hh pathway, involving TGF beta/SMAD (transforming growth factor-beta/Sma- and Mad-related family) signaling. Knockdown of GLI1 and GLI2 restored sensitivity to vemurafenib-resistant cells, an effect associated with both growth arrest and senescence. Treatment of vemurafenib-resistant cells with the GLI1/GLI2 inhibitor Gant61 led to decreased invasion of the melanoma cells in a three-dimensional skin reconstruct model and was associated with a decrease in metalloproteinase (MMP2/MMP9) expression and microphthalmia transcription factor upregulation. Gant61 monotherapy did not alter the drug sensitivity of naive cells, but could reverse the resistance of melanoma cells chronically treated with vemurafenib. We further noted that alternating dosing schedules of Gant61 and vemurafenib prevented the onset of BRAFi resistance, suggesting that this could be a potential therapeutic strategy for the prevention of therapeutic escape. Our results suggest that targeting the Hh pathway in BRAFi-resistant melanoma may represent a viable therapeutic strategy to restore vemurafenib sensitivity, reducing or even inhibiting the acquired chemoresistance in melanoma patients.Fapesp-grant number 2012/04194-1, 2013/05172-4, 2014/24400-0 and 2015/10821-7, CNPq-grant number 150447/2013-2 and 471512/2013-3 and PRODOC-grant no 3193-32/2010. Work in the lab of KS Smalley was supported by the National Institutes of Health grants R01 CA161107, R21 CA198550, and Skin SPORE grant P50 CA168536info:eu-repo/semantics/publishedVersio

Toe Pressure and Toe Brachial Index are Predictive of Cardiovascular Mortality, Overall Mortality, and Amputation Free Survival in Patients with Peripheral Artery Disease

Objective/Background: Peripheral haemodynamic parameters are used to assess the presence and severity of peripheral artery disease (PAD). The prognostic value of ankle brachial index (ABI) has been thoroughly delineated. Nonetheless, the relative usefulness of ankle pressure (AP), ABI, toe pressure (TP), and toe brachial index (TBI) in assessing patient outcome has not been investigated in a concurrent study setting. This study aimed to resolve the association of all four non-invasive haemodynamic parameters in clinically symptomatic patients with PAD with cardiovascular mortality, overall mortality, and amputation free survival (AFS). Methods: In total, 732 symptomatic patients with PAD admitted to the Department of Vascular Surgery for conventional angiography at Turku University Hospital, Turku, Finland, between January 2009 and August 2011 were reviewed retrospectively. Demographic factors, cardiovascular mortality, all-cause mortality, and above foot level amputations were obtained and assessed in relation to AP, ABI, TP, and TBI by means of Kaplan-Meier life tables and a multivariate Cox regression model. Results: The haemodynamic parameter that was associated with poor 36 month general outcome was TP <30 mmHg. Univariate Cox regression analysis of stratified values showed that TP and TBI associated significantly with mortality. In multivariate analysis both TP and TBI were associated with a significant risk of death. For TP <30 mmHg and TBI <0.25 the risk of cardiovascular mortality was hazard ratio [HR] 2.84, 95% confidence interval [CI] 1.75-4.61 [p Conclusion: Among non-invasive haemodynamic measurements and pressure indices both TP and TBI appear to be associated with cardiovascular and overall mortality and AFS for patients with PAD presenting symptoms of the disease. (C) 2017 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.Peer reviewe

Stencil Printing-A Novel Manufacturing Platform for Orodispersible Discs

Stencil printing is a commonly used printing method, but it has not previously been used for production of pharmaceuticals. The aim of this study was to explore whether stencil printing of drug containing polymer inks could be used to manufacture flexible dosage forms with acceptable mass and content uniformity. Formulation development was supported by physicochemical characterization of the inks and final dosage forms. The printing of haloperidol (HAL) discs was performed using a prototype stencil printer. Ink development comprised of investigations of ink rheology in combination with printability assessment. The results show that stencil printing can be used to manufacture HAL doses in the therapeutic treatment range for 6-17 year-old children. The therapeutic HAL dose was achieved for the discs consisting of 16% of hydroxypropyl methylcellulose (HPMC) and 1% of lactic acid (LA). The formulation pH remained above pH 4 and the results imply that the drug was amorphous. Linear dose escalation was achieved by an increase in aperture area of the print pattern, while keeping the stencil thickness fixed. Disintegration times of the orodispersible discs printed with 250 and 500 mu m thick stencils were below 30 s. In conclusion, stencil printing shows potential as a manufacturing method of pharmaceuticals