In Vivo,In Vitro,and In Silico Characterization of Peptoids as Antimicrobial Agents

Bacterial resistance to conventional antibiotics is a global threat that has spurred the development of antimicrobial peptides (AMPs) and their mimetics as novel anti-infective agents. While the bioavailability of AMPs is often reduced due to protease activity, the non-natural structure of AMP mimetics renders them robust to proteolytic degradation, thus offering a distinct advantage for their clinical application. We explore the therapeutic potential of N-substituted glycines, or peptoids, as AMP mimics using a multi-faceted approach that includes in silico, in vitro, and in vivo techniques. We report a new QSAR model that we developed based on 27 diverse peptoid sequences, which accurately correlates antimicrobial peptoid structure with antimicrobial activity. We have identified a number of peptoids that have potent, broad-spectrum in vitro activity against multi-drug resistant bacterial strains. Lastly, using a murine model of invasive S. aureus infection, we demonstrate that one of the best candidate peptoids at 4 mg/kg significantly reduces with a two-log order the bacterial counts compared with saline-treated controls. Taken together, our results demonstrate the promising therapeutic potential of peptoids as antimicrobial agents.     

Acetazolamide Inhibits the Level of Tyrosinase and Melanin: An Enzyme Kinetic,In Vitro,In Vivo,and In Silico Studies

Melanin is the major factor that determines skin color and protects from ultraviolet radiation. In present study we evaluated the anti‐melanogenesis effect of acetazolamide (ACZ) using four different approaches: enzyme kinetic, in vitro, in vivo and in silico. ACZ demonstrated significant inhibitory activity (IC₅₀ 7.895 ± 0.24 μm ) against tyrosinase as compared to the standard drug kojic acid (IC₅₀ 16.84 ± 0.64 μm ) and kinetic analyses showed that ACZ is a non‐competitive inhibitor without cytotoxic effect. In in vitro experiments, A375 human melanoma cells were treated with 20 or 40 μm of ACZ with or without 50 μm of l ‐DOPA. Western blot results showed that ACZ significantly (P < 0.05) decreased the expression level of tyrosinase at 40 μm . Zebrafish embryos were treated with 10, 20 or 40 μm of ACZ and of positive control kojic acid. At 72 h of treatment with ACZ and kojic acid, ACZ significantly (P < 0.001) decreased the embryos pigmentation to 40.8% of untreated embryos at the dose of 40 μm of ACZ while kojic acid decreased only 25.0% of pigmentation at the same dose of kojic acid. In silico docking were performed against tyrosinase using PyRx tool. Docking studies suggested that His244, Asn260 and His85 are the major interacting residues in the binding site of the protein. In conclusion, our results suggest that ACZ is a good candidate for the inhibition of melanin and it could be used as a lead for developing the drugs for hyperpigmentary disorders and skin whitening.     

Combined In Silico,In Vivo,and In Vitro Studies Shed Insights into the Acute Inflammatory Response in Middle-Aged Mice

We combined in silico, in vivo, and in vitro studies to gain insights into age-dependent changes in acute inflammation in response to bacterial endotoxin (LPS). Time-course cytokine, chemokine, and NO₂ ^−/NO₃ ⁻ data from “middle-aged” (6–8 months old) C57BL/6 mice were used to re-parameterize a mechanistic mathematical model of acute inflammation originally calibrated for “young” (2–3 months old) mice. These studies suggested that macrophages from middle-aged mice are more susceptible to cell death, as well as producing higher levels of pro-inflammatory cytokines, vs. macrophages from young mice. In support of the in silico-derived hypotheses, resident peritoneal cells from endotoxemic middle-aged mice exhibited reduced viability and produced elevated levels of TNF-α, IL-6, IL-10, and KC/CXCL1 as compared to cells from young mice. Our studies demonstrate the utility of a combined in silico, in vivo, and in vitro approach to the study of acute inflammation in shock states, and suggest hypotheses with regard to the changes in the cytokine milieu that accompany aging.     

MEK Inhibitors,Novel Anti-Adhesive Molecules,Reduce Sickle Red Blood Cell Adhesion In Vitro and In Vivo,and Vasoocclusion In Vivo

In sickle cell disease, sickle erythrocyte (SSRBC) interacts with endothelial cells, leukocytes, and platelets, and activates coagulation and inflammation, promoting vessel obstruction, which leads to serious life-threatening complications, including acute painful crises and irreversible damage to multiple organs. The mitogen-activated protein kinase, ERK1/2, is abnormally activated in SSRBCs. However, the therapeutic potential of SSRBC ERK1/2 inactivation has never been investigated. I tested four different inhibitors of MEK1/2 (MEK), the kinase that activates ERK1/2, in a model of human SSRBC adhesion to TNFα-activated endothelial cells (ECs). SSRBC MEK inhibition abrogated adhesion to non-activated and TNFα-activated ECs to levels below baseline SSRBC adhesion to non-activated ECs in vitro. SSRBC MEK inhibition also prevented SSRBCs from activating naïve neutrophils to adhere to endothelium. To determine the effect of MEK inhibitors on SSRBC adherence in vivo, sham-treated or MEK inhibitor-treated SSRBCs were infused to nude mice previously treated with TNFα. Sham-treated SSRBCs displayed marked adhesion and occlusion of enflamed vessels, both small and large. However, SSRBC treatment with MEK inhibitors ex vivo showed poor SSRBC adhesion to enflamed vessels with no visible vasoocclusion in vivo. In addition, MEK inhibitor treatment of SSRBCs reduced SSRBC organ trapping and increased the number of SSRBCs circulating in bloodstream. Thus, these data suggest that SSRBC ERK1/2 plays potentially a critical role in sickle pathogenesis, and that MEK inhibitors may represent a valuable intervention for acute sickle cell crises.     

Synthetic Oxoisoaporphine Alkaloids: In Vitro,In Vivo and In Silico Assessment of Antileishmanial Activities

Leishmaniasis is a growing health problem worldwide. As there are certain drawbacks with the drugs currently used to treat human leishmaniasis and resistance to these drugs is emerging, there is a need to develop novel antileishmanial compounds, among which isoquinoline alkaloids are promising candidates. In this study, 18 novel oxoisoaporphine derivatives were synthesized and their possible antileishmanial activity was evaluated. The in vitro activity of these derivatives against Leishmania amazonensis axenic amastigotes was first evaluated, and the selected compounds were then tested in an inhibition assay with promastigotes of L. infantum, L. braziliensis, L. amazonensis and L. guyanensis, and with intracellular amastigotes of L. infantum and L. amazonensis. Finally, the most active compounds, OXO 1 (2,3-dihydro-7H-dibenzo[de,h]quinolin-7-one) and OXO 13 (2,3,8,9,10,11-hexahydro-7H-dibenzo[de,h]quinolin-7-one), were tested in BALB/c mice infected with L. infantum. Treatment of mice at a dose of 10 mg/kg with OXO 1 yielded significant reductions (p<0.05) in parasite burden in liver and spleen (99% and 78%, respectively) whereas with OXO 13 were not significant. Although previous reports suggest that this family of molecules displays inhibitory activity against monoamine oxidase A and acetylcholinesterase, these enzymes were not confirmed as targets for antileishmanial activity on the basis of the present results. However, after development of a new bioinformatics model to analyze the Leishmania proteome, we were able to identify other putative targets for these molecules. The most promising candidates were four proteins: two putative pteridine reductase 2 (1MXF and 1MXH), one N-myristoyltransferase (2WUU) and one type I topoisomerase (2B9S).     

Custom In The Courtroom, Law In The Village: Legal Transformations In Papua New Guinea

After more than twenty-five years on the legal landscape of Papua New Guinea, 'customary law' is ripe for reassessment, particularly as it appears to be an ideal mechanism with which the Papua New Guinean state can meet some of its obligations to a burgeoning body of international law. This article addresses the need to understand customary law in the context of its varying usage across different legal domains in an archetypally pluralistic state. In contrast to older approaches focusing on the problematic interface between an exogenous legal system and indigenous methods of dispute settlement, my concern is with the ways in which these distinct legal forms have fared in each other's company since independence in 1975. Case-studies from a village court and an urban national court demonstrate that village court magistrates and high court judges alike use custom and law as strategic sources of authority. While village courts take custom for granted and therefore must 'discover' law, high courts take law for granted and must 'discover' custom. These processes indicate that, rather than being hybridized as 'customary law', the distinctiveness of custom and law are often maintained in order for one to appear as a resource upon which the other can draw.     

In Memoriam Mila Rainof, MD

Since 1839, Yale medical students have been writing theses as part of their professional training. It is an introduction to the practice of original research, a demanding and sometimes exhausting pursuit. The thesis project promotes a tenacity well suited for the practice of medicine. The thesis advisor has a challenging role as well — one that can only be filled by an individual whose dedication to research is matched with a patience for mentoring students.     

Armen Galoyan,In Memoriam…

Neurochemical Research -     

Combined In Situ Zymography, Immunofluorescence, And Staining Of Iron Oxide Particles In Paraffin-embedded, Zinc-fixed Tissue Sections

AbstractSuperparamagnetic iron oxide particles are used as potent contrast agents in magnetic resonance imaging. In histology, these particles are frequently visualized by Prussian blue iron staining of aldehyde-fixed, paraffin-embedded tissues. Recently, zinc salt-based fixative was shown to preserve enzyme activity in paraffin-embedded tissues. In this study, we demonstrate that zinc fixation allows combining in situ zymography with fluorescence immunohistochemistry (IHC) and iron staining for advanced biologic investigation of iron oxide particle accumulation. Very small iron oxide particles, developed for magnetic resonance angiography, were applied intravenously to BALB/c nude mice. After 3 hours, spleens were explanted and subjected to zinc fixation and paraffin embedding. Cut tissue sections were further processed to in situ zymography, IHC, and Prussian blue staining procedures. The combination of in situ zymography as well as IHC with subsequent Prussian blue iron staining on zinc-fixed paraffin-embedded tissues resulted in excellent histologic images of enzyme activity, protease distribution, and iron oxide particle accumulation. The combination of all three stains on a single section allowed direct comparison with only moderate degradation of fluorescein isothiocyanate-labeled substrate. This protocol is useful for investigating the biologic environment of accumulating iron oxide particles, with excellent preservation of morphology.     

Preliminary Studies with a New Hypoxia-Selective Cytotoxin, KS119W, In Vitro and In Vivo

Agents with selective toxicity to hypoxic cells have shown promise as adjuncts to radiotherapy. Our previous studies showed that the bioreductive alkylating agent KS119 had an extremely large differential toxicity to severely hypoxic and aerobic cells in cell culture, and was effective in killing the hypoxic cells of EMT6 mouse mammary tumors in vivo. However, the limited solubility of that compound precluded its development as an anticancer drug. Here we report our initial studies with KS119W, a water-soluble analog of KS119. The cytotoxicity of KS119W to EMT6 cells in vitro was similar to that of KS119, with both agents producing only minimal cytotoxicity to aerobic cells even after intensive treatments, while producing pronounced cytotoxicity to oxygen-deficient cells. This resulted in large differentials in the toxicities to hypoxic and aerobic cells (>1,000-fold at 10 μM). Low pH had only minimal effects on the cytotoxicity of KS119W. Under hypoxic conditions, EMT6 cells transfected to express high levels of either human or mouse versions of the repair protein O(6)-alkylguanine-DNA alkyltransferase, which is also known as O(6)-methylguanine DNA-methyltransferase, were much more resistant to KS119W than parental EMT6 cells lacking O(6)-alkylguanine-DNA alkyltransferase, confirming the importance of DNA O-6-alkylation to the cytotoxicity of this agent. Studies with EMT6 tumors in BALB/c Rw mice using both tumor cell survival and tumor growth delay assays showed that KS119W was effective as an adjunct to irradiation for the treatment of solid tumors in vivo, producing additive or supra-additive effects in most combination regimens for which the interactions could be evaluated. Our findings encourage additional preclinical studies to examine further the antineoplastic effects of KS119W alone and in combination with radiation, and to examine the pharmacology and toxicology of this new bioreductive alkylating agent so that its potential for clinical use as an adjuvant to radiotherapy can be evaluated.     

Nanosuspension Based In Situ Gelling Nasal Spray of Carvedilol: Development, In Vitro and In Vivo Characterization

The objective of the present investigation was to develop in situ gelling nasal spray formulation of carvedilol (CRV) nanosuspension to improve the bioavailability and therapeutic efficiency. Solvent precipitation–ultrasonication method was opted for the preparation of CRV nanosuspension which further incorporated into the in situ gelling polymer phase. Optimized formulation was extensively characterized for various physical parameters like in situ gelation, rheological properties and in vitro drug release. Formation of in situ gel upon contact with nasal fluid was conferred via the use of ion-activated gellan gum as carrier. In vivo studies in rabbits were performed comparing the nasal bioavailability of CRV after oral, nasal, and intravenous administration. Optimized CRV nanosuspension prepared by combination of poloxamer 407 and oleic acid showed good particle size [d (0.9); 0.19 μm], zeta potential (+10.2 mV) and polydispersity (span; 0.63). The formulation containing 0.5% w/v gellan gum demonstrated good gelation ability and desired sustained drug release over period of 12 h. In vivo pharmacokinetic study revealed that the absolute bioavailability of in situ nasal spray formulation (69.38%) was significantly increased as compared to orally administered CRV (25.96%) with mean residence time 8.65 h. Hence, such in situ gel system containing drug nanosuspension is a promising approach for the intranasal delivery in order to increase nasal mucosal permeability and in vivo residence time which altogether improves drug bioavailability.KEY WORDS: bioavailability, Carvedilol, in situ gel, intranasal, nanosuspension     

Fibronectins,Their Fibrillogenesis,and In Vivo Functions

Fibronectin (FN) is a multidomain protein with the ability to bind simultaneously to cell surface receptors, collagen, proteoglycans, and other FN molecules. Many of these domains and interactions are also involved in the assembly of FN dimers into a multimeric fibrillar matrix. When, where, and how FN binds to its various partners must be controlled and coordinated during fibrillogenesis. Steps in the process of FN fibrillogenesis including FN self-association, receptor activities, and intracellular pathways have been under intense investigation for years. In this review, the domain organization of FN including the extra domains and variable region that are controlled by alternative splicing are described. We discuss how FN–FN and cell–FN interactions play essential roles in the initiation and progression of matrix assembly using complementary results from cell culture and embryonic model systems that have enhanced our understanding of this process.As a ubiquitous component of the extracellular matrix (ECM), fibronectin (FN) provides essential connections to cells through integrins and other receptors and regulates cell adhesion, migration, and differentiation. FN is secreted as a large dimeric glycoprotein with subunits that range in size from 230 kDa to 270 kDa (Mosher 1989; Hynes 1990). Variation in subunit size depends primarily on alternative splicing. FN was first isolated from blood more than 60 years ago (Edsall 1978), and this form is called plasma FN. The other major form, called cellular FN, is abundant in the fibrillar matrices of most tissues. Although FN is probably best known for promoting attachment of cells to surfaces, this multidomain protein has many interesting structural features and functional roles beyond cell adhesion.FN is composed of three different types of modules termed type I, II, and III repeats (Fig. 1) (Petersen et al. 1983; Hynes 1990). These repeats have distinct structures. Although the conformations of type I and type II repeats are maintained by pairs of intramodule disulfide bonds, the type III repeat is a 7-stranded β-barrel structure that lacks disulfide bonds (Main et al. 1992; Leahy et al. 1996, 1992) and, therefore, can undergo conformational changes. FN type III repeats are widely distributed among animal, bacterial, and plant proteins and are found in both extracellular and intracellular proteins (Bork and Doolittle 1992; Tsyguelnaia and Doolittle 1998).Open in a separate windowFigure 1.FN domain organization and isoforms. Each FN monomer has a modular structure consisting of 12 type I repeats (cylinders), 2 type II repeats (diamonds), and 15 constitutive type III repeats (hexagons). Two additional type III repeats (EIIIA and EIIIB, green) are included or omitted by alternative splicing. The third region of alternative splicing, the V region (green box), is included (V120), excluded (V0), or partially included (V95, V64, V89). Sets of modules comprise domains for binding to other extracellular molecules as indicated. Domains required for fibrillogenesis are in red: the assembly domain (repeats I_1-5) binds FN, III_9-10 contains the RGD and synergy sequences for integrin binding, and the carboxy-terminal cysteines form the disulfide-bonded FN dimer (‖). The III_1-2 domain (light red) has two FN binding sites that are important for fibrillogenesis. The amino-terminal 70-kDa fragment contains assembly and gelatin-binding domains and is routinely used in FN binding and matrix assembly studies.Sets of adjacent modules form binding domains for a variety of proteins and carbohydrates (Fig. 1). ECM proteins, including FN, bind to cells via integrin receptors, αβ heterodimers with two transmembrane subunits (Hynes 2002). FN-binding integrins have specificity for one of the two cell-binding sites within FN, either the RGD-dependent cell-binding domain in III₁₀ (Pierschbacher and Ruoslahti 1984) or the CS1 segment of the alternatively spliced V region (IIICS) (Wayner et al. 1989; Guan and Hynes 1990). Some integrins require a synergy sequence in repeat III₉ for maximal interactions with FN (Aota et al. 1994; Bowditch et al. 1994). Another family of cell surface receptors is the syndecans, single-chain transmembrane proteoglycans (Couchman 2010). Syndecans use their glycosaminoglycan (GAG) chains to interact with FN at its carboxy-terminal heparin-binding (HepII) domain (Fig. 1) (Saunders and Bernfield 1988; Woods et al. 2000), which binds to heparin, heparan sulfate, and chondroitin sulfate GAGs (Hynes 1990; Barkalow and Schwarzbauer 1994). Syndecan binding to the HepII domain enhances integrin-mediated cell spreading and intracellular signaling, suggesting that syndecans act as coreceptors with integrins in cell–FN binding (Woods and Couchman 1998; Morgan et al. 2007).A major site for FN self-association is within the amino-terminal assembly domain spanning the first five type I repeats (I_1-5) (Fig. 1) (McKeown-Longo and Mosher 1985; McDonald et al. 1987; Schwarzbauer 1991b; Sottile et al. 1991). This domain plays an essential role in FN fibrillogenesis. As a major blood protein, FN interacts with fibrin during blood coagulation, also using the I_1-5 domain (Mosher 1989; Hynes 1990). As fibrin polymerizes, factor XIII transglutaminase covalently cross-links glutamine residues near the amino terminus of FN to fibrin α chains (Mosher 1975; Corbett et al. 1997). The amino-terminal domain has multiple binding partners in addition to FN and fibrin; these include heparin, S. aureus, and other bacteria, thrombospondin-1, and tenascin-C (Hynes 1990; Ingham et al. 2004; Schwarz-Linek et al. 2006). Adjacent to this domain is the gelatin/collagen-binding domain composed of type I and type II modules (Ingham et al. 1988). This domain also binds to tissue transglutaminase (Radek et al. 1993) and fibrillin-1 (Sabatier et al. 2009). Within the 15 type III repeats reside several FN binding sites that interact with the amino-terminal assembly domain as well as three sites of alternative splicing that generate multiple isoforms. At the carboxyl terminus is a pair of cysteine residues that form the FN dimer through antiparallel disulfide bonds (Hynes 1990). This dimerization may be facilitated by disulfide isomerase activity located in the last set of type I repeats (Langenbach and Sottile 1999).The diverse set of binding domains provides FN with the ability to interact simultaneously with other FN molecules, other ECM components (e.g., collagens and proteoglycans), cell surface receptors, and extracellular enzymes (Pankov and Yamada 2002; Fogelgren et al. 2005; Hynes 2009; Singh et al. 2010). Multitasking by FN probably underlies its essential role during embryogenesis (George et al. 1993). Furthermore, FN''s interactions can be modulated by exposure or sequestration of its binding sites within matrix fibrils, through the presence of ECM proteins that bind to FN, or through variation in structure by alternative splicing.     

Radical Formation and Accumulation In Vivo,In Desiccation Tolerant and Intolerant Mosses

Water loss in a desiccation-sensitive moss resulted in destruction of chlorophyll, loss of carotenoids and increased lipid peroxidation, indicating the presence of damaging forms of activated oxygen. These effects were exaggerated when the plants were desiccated at high light intensities. During water-deprivation there was a build up of a free radical, detected in vivo, with a close correlation between molecular damage and radical accumulation. In contrast, in a desiccation-tolerant moss there was almost no indication of molecular (oxidative) damage. However a stable radical similar in type and concentration to that found in the desiccation-sensitive species accumulated, particularly under high irradiances. The stable radical appears to be one of the end-products of a process initiated by environmental stress, desiccation and high irradiance: its association with molecular damage depending on the degree to which the species is tolerant of desiccation. Identification of the radical in intact tissue from EPR and ENDOR studies, suggests that this is not a short-lived proxy-radical but instead is relatively stable and carbon-centred.     

Radical Formation and Accumulation In Vivo, In Desiccation Tolerant and Intolerant Mosses

Water loss in a desiccation-sensitive moss resulted in destruction of chlorophyll, loss of carotenoids and increased lipid peroxidation, indicating the presence of damaging forms of activated oxygen. These effects were exaggerated when the plants were desiccated at high light intensities. During water-deprivation there was a build up of a free radical, detected in vivo, with a close correlation between molecular damage and radical accumulation. In contrast, in a desiccation-tolerant moss there was almost no indication of molecular (oxidative) damage. However a stable radical similar in type and concentration to that found in the desiccation-sensitive species accumulated, particularly under high irradiances. The stable radical appears to be one of the end-products of a process initiated by environmental stress, desiccation and high irradiance: its association with molecular damage depending on the degree to which the species is tolerant of desiccation. Identification of the radical in intact tissue from EPR and ENDOR studies, suggests that this is not a short-lived proxy-radical but instead is relatively stable and carbon-centred.