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In the global effort to thwart antimicrobial resistance, lipopeptides are an important class of antimicrobial agents, especially against Gram-negative infections. In an attempt to circumvent their synthetic complexities, we designed simple membrane-active agents involving only one amino acid and two lipid tails. Herein we show that the use of two short lipid tails instead of a single long one significantly increases selective antibacterial activity. This study yielded several selective antibacterial compounds, and investigations into the properties of this compound class were conducted with the most active compound. Fluorescence spectroscopic studies revealed the capacity of the representative compound to cause depolarization and permeabilization of bacterial cell membranes. This membrane-active nature of the compound imparts superior activity against persister cells, biofilms, and planktonic cells. Topical application of the compound decreased bacterial burden in mice inflicted with burn-infections caused by Acinetobacter baumannii. We anticipate that the design principles described herein will direct the development of several antimicrobial agents of clinical importance., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The continuous rise of antimicrobial resistance and the dearth of new antibiotics in the clinical pipeline raise an urgent call for the development of potent antimicrobial agents. Cationic chitosan derivatives, N-(2-hydroxypropyl)-3-trimethylammonium chitosan chlorides (HTCC), have been widely studied as potent antibacterial agents. However, their systemic structure-activity relationship, activity toward drug-resistant bacteria and fungi, and mode of action are very rare. Moreover, toxicity and efficacy of these polymers under in vivo conditions are yet to be established. Herein, we investigated antibacterial and antifungal efficacies of the HTCC polymers against multidrug resistant bacteria including clinical isolates and pathogenic fungi, studied their mechanism of action, and evaluated cytotoxic and antimicrobial activities in vitro and in vivo. The polymers were found to be active against both bacteria and fungi (MIC = 125-250 μg/mL) and displayed rapid microbicidal kinetics, killing pathogens within 60-120 min. Moreover, the polymers were shown to target both bacterial and fungal cell membrane leading to membrane disruption and found to be effective in hindering bacterial resistance development. Importantly, very low toxicity toward human erythrocytes (HC 50 = >10000 μg/mL) and embryo kidney cells were observed for the cationic polymers in vitro. Further, no inflammation toward skin tissue was observed in vivo for the most active polymer even at 200 mg/kg when applied on the mice skin. In a murine model of superficial skin infection, the polymer showed significant reduction of methicillin-resistant Staphylococcus aureus (MRSA) burden (3.2 log MRSA reduction at 100 mg/kg) with no to minimal inflammation. Taken together, these selectively active polymers show promise to be used as potent antimicrobial agents in topical and other infections.

Cationic-amphiphilic antibacterial polymers with optimal amphiphilicity generally target the bacterial membranes instead of mammalian membranes. To date, this balance has been achieved by varying the cationic charge or side chain hydrophobicity in a variety of cationic-amphiphilic polymers. Optimal hydrophobicity of cationic-amphiphilic polymers has been considered as the governing factor for potent antibacterial activity yet minimal mammalian cell toxicity. However, the concomitant role of hydrogen bonding and hydrophobicity with constant cationic charge in the interactions of antibacterial polymers with bacterial membranes is not understood. Also, degradable polymers that result in nontoxic degradation byproducts offer promise as safe antibacterial agents. Here we show that amide- and ester (degradable)-bearing cationic-amphiphilic polymers with tunable side chain hydrophobicity can modulate antibacterial activity and cytotoxicity. Our results suggest that an amide polymer can be a potent antibacterial agent with lower hydrophobicity whereas the corresponding ester polymer needs a relatively higher hydrophobicity to be as effective as its amide counterpart. Our studies reveal that at higher hydrophobicities both amide and ester polymers have similar profiles of membrane-active antibacterial activity and mammalian cell toxicity. On the contrary, at lower hydrophobicities, amide and ester polymers are less cytotoxic, but the former have potent antibacterial and membrane activity compared to the latter. Incorporation of amide and ester moieties made these polymers side chain degradable, with amide polymers being more stable than the ester polymers. Further, the polymers are less toxic, and their degradation byproducts are nontoxic to mice. More importantly, the optimized amide polymer reduces the bacterial burden of burn wound infections in mice models. Our design introduces a new strategy of interplay between the hydrophobic and hydrogen bonding interactions keeping constant cationic charge density for developing potent membrane-active antibacterial polymers with minimal toxicity to mammalian cells.

Correction for 'Selective and broad spectrum amphiphilic small molecules to combat bacterial resistance and eradicate biofilms' by Jiaul Hoque et al., Chem. Commun., 2015, 51, 13670-13673.

If surface effects are neglected, any change of the Fermi level in a semiconductor is expected to result in an equal and opposite change of the work function. However, this is in general not observed in three-dimensional semiconductors, because of Fermi level pinning at the surface. By combining Kelvin probe force microscopy and scanning tunneling spectroscopy on single layer graphene, we measure both the local work function and the charge carrier density. The one-to-one equivalence of changes in the Fermi level and the work function is demonstrated to accurately hold in single layer graphene down to the nanometer scale.

Biomimetic antibacterial polymers, the functional mimics of antimicrobial peptides (AMPs), targeting the bacterial cell membrane have been developed to combat the problem of antibiotic resistance. Amphiphilicity, a balance of cationic charge and hydrophobicity, in these polymers has been shown to be pivotal for their selective interactions with anionic lipid membranes of bacteria instead of zwitterionic mammalian (human erythrocyte) membranes. However, it is unclear if and to what extent hydrogen bonding in amphiphilic antibacterial polymers contributes to this membrane binding specificity. To address this, we employ isosteric substitution of ester with amide moieties that differ in their potency for hydrogen bonding in the side chains of N -alkyl maleimide based amphiphilic polymers. Our studies reveal that amide polymer (AC3P) is a potent antibacterial agent with high membrane-disrupting properties compared to its ester counterpart (EC3P). To understand these differences we performed bio-physical experiments and molecular dynamics (MD) simulations which showed strong interactions of AC3P including hydrogen bonding with lipid head groups of bacterial model lipid bilayers, that are absent in EC3P, make them selective for bacterial membranes. Mechanistic investigations of these polymers in bacteria revealed specific membrane disruptive activity leading to the delocalization of cell division related proteins. This unprecedented and unique concept provides an understanding of bacterial membrane interactions highlighting the role of hydrogen bonding. Thus, these findings will have significant implications in efficient design of potent membrane-active agents.

Vancomycin, the drug of last resort for Gram-positive bacterial infections, has also been rendered ineffective by the emergence of resistance in such bacteria. To combat the threat of vancomycin-resistant bacteria (VRB), we report the development of a dipicolyl-vancomycin conjugate (Dipi-van), which leads to enhanced inhibition of cell-wall biosynthesis in VRB and displays in vitro activity that is more than two orders of magnitude higher than that of vancomycin. Conjugation of the dipicolyl moiety, which is a zinc-binding ligand, endowed the parent drug with the ability to bind to pyrophosphate groups of cell-wall lipids while maintaining the inherent binding affinity for pentapeptide termini of cell-wall precursors. Furthermore, no detectable resistance was observed after several serial passages, and the compound reduced the bacterial burden by a factor of 5 logs at 12 mg kg(-1) in a murine model of VRB kidney infection. The findings presented in this report stress the potential of our strategy to combat VRB infections., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Staphylococcus aureus is a facultative intracellular pathogen and there are limited options for the treatment of severe intracellular bacterial infections. The membrane-active glycopeptide antibiotic Van-QC8 is a permanent positively charged lipophilic vancomycin analogue that demonstrates high activity against clinically relevant drug-resistant Gram-positive bacteria both in vitro and in vivo. In this study, the intracellular activity of Van-QC8 was evaluated against meticillin-resistant S. aureus (MRSA) infection in RAW macrophages. Furthermore, the mechanism of intracellular uptake of Van-QC8 was investigated. Van-QC8 showed time- and concentration-dependent bactericidal activity against intracellular MRSA. Van-QC8 displayed significantly higher intracellular activity compared with vancomycin and linezolid. Cellular uptake of Van-QC8 was found to be through clathrin-dependent and -independent and caveolin-dependent and -independent endocytic pathways. The findings of this study suggest that Van-QC8 could be translated clinically for the treatment of intracellular infections due to MRSA., (Copyright © 2016 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

Symbiosis receptor kinase (SYMRK) is indispensable for activation of root nodule symbiosis (RNS) at both epidermal and cortical levels and is functionally conserved in legumes. Previously, we reported SYMRK to be phosphorylated on "gatekeeper" Tyr both in vitro as well as in planta. Since gatekeeper phosphorylation was not necessary for activity, the significance remained elusive. Herein, we show that substituting gatekeeper with nonphosphorylatable residues like Phe or Ala significantly affected autophosphorylation on selected targets on activation segment/αEF and β3-αC loop of SYMRK. In addition, the same gatekeeper mutants failed to restore proper symbiotic features in a symrk null mutant where rhizobial invasion of the epidermis and nodule organogenesis was unaffected but rhizobia remain restricted to the epidermis in infection threads migrating parallel to the longitudinal axis of the root, resulting in extensive infection patches at the nodule apex. Thus, gatekeeper phosphorylation is critical for synchronizing epidermal/cortical responses in RNS., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Membrane-active agents represent a promising alternative to overcome antibiotic resistance. Here, we report cationic-amphiphilic polymers with variations in the side chain architecture such as cyclization, isomerization and unsaturation that resulted in potent antibacterial activity and low mammalian cell toxicity with a membrane-active mode of action.

The charge carrier density in graphene on a dielectric substrate such as SiO_{2} displays inhomogeneities, the so-called charge puddles. Because of the linear dispersion relation in monolayer graphene, the puddles are predicted to grow near charge neutrality, a markedly distinct property from conventional two-dimensional electron gases. By performing scanning tunneling microscopy and spectroscopy on a mesoscopic graphene device, we directly observe the puddles' growth, both in spatial extent and in amplitude, as the Fermi level approaches the Dirac point. Self-consistent screening theory provides a unified description of both the macroscopic transport properties and the microscopically observed charge disorder.

Bacterial biofilms represent the root-cause of chronic or persistent infections in humans. Gram-negative bacterial infections due to nosocomial and opportunistic pathogens such as Acinetobacter baumannii are more difficult to treat because of their inherent and rapidly acquiring resistance to antibiotics. Due to biofilm formation, A. baumannii has been noted for its apparent ability to survive on artificial surfaces for an extended period of time, therefore allowing it to persist in the hospital environment. Here we report, maleic anhydride based novel cationic polymers appended with amide side chains that disrupt surface established multi-drug resistant A. baumannii biofilms. More importantly, these polymers significantly (p < 0.0001) decrease the bacterial burden in mice with chronic A. baumannii burn wound infection. The polymers also show potent antibacterial efficacy against methicillin resistant Staphylococcus aureus (MRSA), vancomycin resistant Enterococci (VRE) and multi-drug resistant clinical isolates of A. baumannii with minimal toxicity to mammalian cells. We observe that optimal hydrophobicity dependent on the side chain chemical structure of these polymers dictate the selective toxicity to bacteria. Polymers interact with the bacterial cell membranes by causing membrane depolarization, permeabilization and energy depletion. Bacteria develop rapid resistance to erythromycin and colistin whereas no detectable development of resistance occurs against these polymers even after several passages. These results suggest the potential use of these polymeric biomaterials in disinfecting biomedical device surfaces after the infection has become established and also for the topical treatment of chronic bacterial infections., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Spinal cord injury (SCI) has become one of the most leading concerns in the past decade. Preclinical and research studies are now ongoing trying to understand the molecular mechanisms and develop treatment strategies for this neurodegenerative condition. In the last decade, researchers have deciphered few of the leading players that play a major role in worsening the condition. But till date none of these have been applied to the clinical treatment of patients with SCI. Here in this chapter I discuss about one of the dietary requirements that could ameliorate the condition of these patients.

The emergence of bacterial resistance and biofilm associated infections has created a challenging situation in global health. In this present state of affairs where conventional antibiotics are falling short of being able to provide a solution to these problems, development of novel antibacterial compounds possessing the twin prowess of antibacterial and antibiofilm efficacy is imperative. Herein, we report a library of amino acid tunable lipidated norspermidine conjugates that were prepared by conjugating both amino acids and fatty acids with the amine functionalities of norspermidine through amide bond formation. These lipidated conjugates displayed potent antibacterial activity against various planktonic Gram-positive and Gram-negative bacteria including drug-resistant superbugs such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, and β-lactam-resistant Klebsiella pneumoniae. This class of nontoxic and fast-acting antibacterial molecules (capable of killing bacteria within 15 min) did not allow bacteria to develop resistance against them after several passages. Most importantly, an optimized compound in the series was also capable of killing metabolically inactive persisters and stationary phase bacteria. Additionally, this compound was capable of disrupting the preformed biofilms of S. aureus and E. coli. Therefore, this class of antibacterial conjugates have potential in tackling the challenging situation posed by both bacterial resistance as well as drug tolerance due to biofilm formation.

Resistance to glycopeptide antibiotics, the drugs of choice for life-threatening bacterial infections, is on the rise. In order to counter the threat of glycopeptide-resistant bacteria, we report development of a new class of semi-synthetic glycopeptide antibiotics, which not only target the bacterial membrane but also display enhanced inhibition of cell-wall biosynthesis through increased binding affinity to their target peptides. The combined effect of these two mechanisms resulted in improved in vitro activity of two to three orders of magnitude over vancomycin and no propensity to trigger drug resistance in bacteria. In murine model of kidney infection, the optimized compound was able to bring bacterial burden down by about 6 logs at 12 mg kg(-1) with no observed toxicity. The results furnished in this report emphasize the potential of this class of compounds as future antibiotics for drug-resistant Gram-positive infections., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Unlabelled: Mycobacterium species such as M. smegmatis and M. tuberculosis encode at least two translesion synthesis (TLS) polymerases, DinB1 and DinB2, respectively. Although predicted to be linked to DNA repair, their role in vivo remains enigmatic. M. smegmatis mc(2)155, a strain commonly used to investigate mycobacterial genetics, has two copies of dinB2, the gene that codes for DinB2, by virtue of a 56-kb chromosomal duplication. Expression of a mycobacteriophage D29 gene (gene 50) encoding a class II ribonucleotide reductase in M. smegmatis ΔDRKIN, a strain derived from mc(2)155 in which one copy of the duplication is lost, resulted in DNA replication defects and growth inhibition. The inhibitory effect could be linked to the deficiency of dTTP that resulted under these circumstances. The selective inhibition observed in the ΔDRKIN strain was found to be due solely to a reduced dosage of dinB2 in this strain. Mycobacterium bovis, which is closely related to M. tuberculosis, the tuberculosis pathogen, was found to be highly susceptible to gene 50 overexpression. Incidentally, these slow-growing pathogens harbor one copy of dinB2. The results indicate that the induction of a dTTP-limiting state can lead to growth inhibition in mycobacteria, with the effect being maximum in cells deficient in DinB2., Importance: Mycobacterium species, such as M. tuberculosis, the tuberculosis pathogen, are known to encode several Y family DNA polymerases, one of which is DinB2, an ortholog of the DNA repair-related protein DinP of Escherichia coli. Although this protein has been biochemically characterized previously and found to be capable of translesion synthesis in vitro, its in vivo function remains unknown. Using a novel method to induce dTTP deficiency in mycobacteria, we demonstrate that DinB2 can aid mycobacterial survival under such conditions. Apart from unraveling a specific role for the mycobacterial Y family DNA polymerase DinB2 for the first time, this study also paves the way for the development of drugs that can kill mycobacteria by inducing a dTTP-deficient state., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Mycobacteriophages infect mycobacteria, resulting in their death. Therefore, the possibility of using them as therapeutic agents against the deadly mycobacterial disease tuberculosis (TB) is of great interest. To obtain better insight into the dynamics of mycobacterial inactivation by mycobacteriophages, this study was initiated using mycobacteriophage D29 and Mycobacterium smegmatis as the phage-host system. Here, we implemented a goal-oriented iterative cycle of experiments on one hand and mathematical modeling combined with Monte Carlo simulations on the other. This integrative approach lends valuable insight into the detailed kinetics of bacterium-phage interactions. We measured time-dependent changes in host viability during the growth of phage D29 in M. smegmatis at different multiplicities of infection (MOI). The predictions emerging out of theoretical analyses were further examined using biochemical and cell biological assays. In a phage-host interaction system where multiple rounds of infection are allowed to take place, cell counts drop more rapidly than expected if cell lysis is considered the only mechanism for cell death. The phenomenon could be explained by considering a secondary factor for cell death in addition to lysis. Further investigations reveal that phage infection leads to the increased production of superoxide radicals, which appears to be the secondary factor. Therefore, mycobacteriophage D29 can function as an effective antimycobacterial agent, the killing potential of which may be amplified through secondary mechanisms., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Rationally designed amphiphilic small molecules selectively kill drug-sensitive and drug-resistant bacteria over mammalian cells. The small molecules disperse preformed biofilms and reduce viable bacterial count in the biofilms. Moreover, this class of membrane-active molecules disarms the development of bacterial resistance.

Copy number variation (CNV) is a form of structural alteration in the mammalian DNA sequence, which are associated with many complex neurological diseases as well as cancer. The development of next generation sequencing (NGS) technology provides us a new dimension towards detection of genomic locations with copy number variations. Here we develop an algorithm for detecting CNVs, which is based on depth of coverage data generated by NGS technology. In this work, we have used a novel way to represent the read count data as a two dimensional geometrical point. A key aspect of detecting the regions with CNVs, is to devise a proper segmentation algorithm that will distinguish the genomic locations having a significant difference in read count data. We have designed a new segmentation approach in this context, using convex hull algorithm on the geometrical representation of read count data. To our knowledge, most algorithms have used a single distribution model of read count data, but here in our approach, we have considered the read count data to follow two different distribution models independently, which adds to the robustness of detection of CNVs. In addition, our algorithm calls CNVs based on the multiple sample analysis approach resulting in a low false discovery rate with high precision.

Treating bacterial biofilms with conventional antibiotics is limited due to ineffectiveness of the drugs and higher propensity to develop bacterial resistance. Development of new classes of antibacterial therapeutics with alternative mechanisms of action has become imperative. Herein, we report the design, synthesis, and biological evaluations of novel membrane-active small molecules featuring two positive charges, four nonpeptidic amide groups, and variable hydrophobic/hydrophilic (amphiphilic) character. The biocides synthesized via a facile methodology not only displayed good antibacterial activity against wild-type bacteria but also showed high activity against various drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), and β-lactam-resistant Klebsiella pneumoniae. Further, these biocides not only inhibited the formation of biofilms but also disrupted the established S. aureus and E. coli biofilms. The membrane-active biocides hindered the propensity to develop bacterial resistance. Moreover, the biocides showed negligible toxicity against mammalian cells and thus bear potential to be used as therapeutic agents.

Natural and synthetic membrane active antibacterial agents offer hope as potential solutions to the problem of bacterial resistance as the membrane-active nature imparts low propensity for the development of resistance. In this report norspermidine based antibacterial molecules were developed that displayed excellent antibacterial activity against various wild-type bacteria (Gram-positive and Gram-negative) and drug-resistant bacteria (methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, and β-lactam-resistant Klebsiella pneumoniae). In a novel structure-activity relationship study it has been shown how incorporation of an aromatic amino acid drastically improves selective antibacterial activity. Additionally, the effect of stereochemistry on activity, toxicity, and plasma stability has also been studied. These rapidly bactericidal, membrane active antibacterial compounds do not trigger development of resistance in bacteria and hence bear immense potential as therapeutic agents to tackle multidrug resistant bacterial infections.

The advanced functionality of portable devices such as smart phones provides the necessary hardware to potentially perform complex diagnostic measurements in any setting. Recent research and development have utilized cameras and data acquisition properties of smart phones to create diagnostic approaches for a variety of diseases or pollutants. However, in concentration measurements, such as blood glucose, the performance of handheld diagnostic devices depends largely on the sensing mechanism. To expand measurements to multiple components, often necessary in medical tests, with a single diagnostic device, robust platform based sensors are needed. Here, we developed a suite of dual wavelength fluorescent sensors with response characteristics necessary to measure each component of a basic metabolic panel, a common clinical measurement. Furthermore, the response of these sensors could be measured with a simple optical setup to convert a smart phone into a fluorescence measurement instrument. This approach could be used as a mobile basic metabolic panel measurement system for point of care diagnostics.

Plant receptor-like kinases (RLKs) are distinguished by having a tyrosine in the 'gatekeeper' position. Previously we reported Symbiosis Receptor Kinase from Arachis hypogaea (AhSYMRK) to autophosphorylate on the gatekeeper tyrosine (Y670), though this phosphorylation was not necessary for the kinase activity. Here we report that recombinant catalytic domain of AhSYMRK with a phosphomimic substitution in the gatekeeper position (Y670E) is catalytically almost inactive and is conformationally quite distinct from the corresponding native enzyme. Additionally, we show that gatekeeper-phosphorylated AhSYMRK polypeptides are inactive and depletion of this inactive form leads to activation of intramolecular autophosphorylation of AhSYMRK. Together, our results suggest gatekeeper tyrosine autophosphorylation to be autoinhibitory for AhSYMRK., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Objectives: To investigate the neuroprotective potential of a saponin isolated from the roots of Momordica cymbalaria against peripheral neuropathy in streptozotocin-induced diabetic rats., Materials and Methods: A steroidal saponin (SMC) was isolated from M. cymbalaria Fenzl and purified by preparative high-performance liquid chromatography. Diabetes was induced in male Wister rats by injecting streptozotocin 45 mg/kg. Diabetic rats were divided into six groups for neuroprotective effect--three each for preventive and curative groups. Neuropathic analgesia was assessed by tail-flick and hot-plate methods. Dorsal root ganglion (DRG) neurons and sciatic nerves were isolated, and histopathological analysis was performed. Antioxidant activity (superoxide dismutase, catalase, and inhibition of lipid peroxidation) of the saponin was also carried out on the isolated DRG neurons and sciatic nerves to assess total oxidative stress., Results: In both preventive and curative protocols, rats administered with SMC showed significant decrease in tail immersion latency time and increase in pain sensitivity when compared to diabetic control group. There was improvement in the myelination and degenerative changes of the nerve fiber in both the groups, and an obvious delay in the progression of neuropathy was evident. SMC treatment showed significant decrease in superoxide dismutase, catalase activity, and lipid peroxidation in the nerves., Conclusions: The steroidal saponin of M. cymbalaria (SMC) possesses potential neuroprotective effect in diabetic peripheral neuropathy with respect to neuropathic analgesia, improvement in neuronal degenerative changes, and significant antioxidant activity.

Glucose uptake by isolated diaphragms of both diabetic, following streptozotocin administration, and non-diabetic animals increased in presence of an oleanane-type triterpenoid saponin isolated from the roots of M. cymbalaria. Insulin release was augmented by the presence of the saponin of M. cymbalaria (1 mg/mL) in rat insulinoma cell line (RIN-5F) preexposed to adrenaline (5 microM) and nifedipine (50 microM). Pancreatic histology also indicated considerable quantitative increase in beta-cells (75%) when treated with the saponin. The results suggest that the saponin of M. cymbalaria possesses potential antidiabetic activity with respect to insulin secretion, which may be attributed to modulation of calcium channel, and beta-cell rejuvenation.

Lipid functionalized single walled carbon nanotube-based self assembly forms a super-micellar structure. This assemblage has been exploited to trap glucose oxidase in a molecular cargo for glucose sensing. The advantage of such a molecular trap is that all components of this unique structure (both the trapping shell and the entrapped enzyme) are reusable and rechargeable. The unique feature of this sensing method lies in the solid state functionalization of single walled carbon nanotubes that facilitates liquid state immobilization of the enzyme. The method can be used for soft-immobilization (a new paradigm in enzyme immobilization) of enzymes with better thermostability that is imparted by the strong hydrophobic environment provided through encapsulation by the nanotubes.

We report a novel method for the fabrication of superconducting nano-devices based on niobium. The well-known difficulties of lithographic patterning of high-quality niobium are overcome by replacing the usual organic resist mask by a metallic one. The quality of the fabrication procedure is demonstrated by the realization and characterization of long and narrow superconducting lines and niobium-gold-niobium proximity SQUIDs.

Plant receptor-like kinases (RLKs) share their evolutionary origin with animal interleukin-1 receptor-associated kinase (IRAK)/Pelle family of soluble kinases and are distinguished by having tyrosine as 'gatekeeper'. This position is adjacent to the hinge region and is hidden in a hydrophobic pocket of the catalytic cleft of protein kinases and is therefore least probable to be a target for any modification. This communication illustrates the accessibility of the gatekeeper site (Y670) towards both autophosphorylation and dephosphorylation in the recombinant cytoplasmic domain of symbiosis receptor kinase from Arachis hypogaea (AhSYMRK). Autophosphorylation on gatekeeper tyrosine was detected prior to extraction but never under in vitro conditions. We hypothesize gatekeeper phosphorylation to be associated with synthesis/maturation of AhSYMRK and this phenomenon may be prevalent among RLKs., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Retroperitoneal amyloidosis has been described in a few case reports and is typically a secondary manifestation of inflammation or malignancy. We present the case of a 69-year-old man who presented with a large pleural effusion and an incidental retroperitoneal mass in the CT imaging. Further investigation confirmed the diagnosis of amyloid amyloidosis involving the retroperitoneum as well as a concurrent Waldenstrom's macroglobulinaemia. Chemotherapy consistent of drugs active against both the lymphoid and plasma cell components of the disease is the proposed therapy for Waldenstrom's macroglobulinaemia in those patients amenable to receive chemotherapy and can make a difference in the survival.

The nature of solvent molecules around proteins in native and different non-native states is crucial for understanding the protein folding problem. We have characterized two compact denatured states of glutaminyl-tRNA synthetase (GlnRS) under equilibrium conditions in the presence of a naturally occurring osmolyte, l-glutamate. The solvation dynamics of the compact denatured states and the fully unfolded state has been studied using a covalently attached probe, acrylodan, near the active site. The solvation dynamics progressively becomes faster as the protein goes from the native to the molten globule to the pre molten globule to the fully unfolded state. Anisotropy decay measurements suggest that the pre-molten-globule intermediate is more flexible than the molten globule although the secondary structure is largely similar. Dynamic light scattering studies reveal that both the compact denatured states are aggregated under the measurement conditions. The implications of solvation dynamics in aggregated compact denatured states have been discussed.

Discrimination between cognate and non-cognate tRNAs by aminoacyl-tRNA synthetases occurs at several steps of the aminoacylation pathway. We have measured changes of solvation and counter-ion distribution at various steps of the aminoacylation pathway of glutamyl- and glutaminyl-tRNA synthetases. The decrease in the association constant with increasing KCl concentration is relatively small for cognate tRNA binding when compared to known DNA-protein interactions. The electro-neutral nature of the tRNA binding domain may be largely responsible for this low ion release stoichiometry, suggesting that a relatively large electrostatic component of the DNA-protein interaction free energy may have evolved for other purposes, such as, target search. Little change in solvation upon tRNA binding is seen. Non-cognate tRNA binding actually increases with increasing KCl concentration indicating that charge repulsion may be a significant component of binding free energy. Thus, electrostatic interactions may have been used to discriminate between cognate and non-cognate tRNAs in the binding step. The catalytic constant of glutaminyl-tRNA synthetase increases with increasing osmotic pressure indicating a water release of 8.4 +/- 1.4 mol/mol in the transition state, whereas little change is seen in the case of glutamyl-tRNA synthetase. We propose that the significant amount of water release in the transition state, in the case of glutaminyl-tRNA synthetase, is due to additional contact of the protein with the tRNA in the transition state.

The urea induced equilibrium denaturation behavior of glutaminyl-tRNA synthetase from Escherichia coli (GlnRS) in 0.25 m potassium l-glutamate, a naturally occurring osmolyte in E. coli, has been studied. Both the native to molten globule and molten globule to unfolded state transitions are shifted significantly toward higher urea concentrations in the presence of l-glutamate, suggesting that l-glutamate has the ability to counteract the denaturing effect of urea. d-Glutamate has a similar effect on the equilibrium denaturation of glutaminyl-tRNA synthetase, indicating that the effect of l-glutamate may not be due to substrate-like binding to the native state. The activation energy of unfolding is not significantly affected in the presence of 0.25 m potassium l-glutamate, indicating that the native state is not preferentially stabilized by the osmolyte. Dramatic increase of coefficient of urea concentration dependence (m) values of both the transitions in the presence of glutamate suggests destabilization and increased solvent exposure of the denatured states. Four other osmolytes, sorbitol, trimethylamine oxide, inositol, and triethylene glycol, show either a modest effect or no effect on native to molten globule transition of glutaminyl-tRNA synthetase. However, glycine betaine significantly shifts the transition to higher urea concentrations. The effect of these osmolytes on other proteins is mixed. For example, glycine betaine counteracts urea denaturation of tubulin but promotes denaturation of S228N lambda-repressor and carbonic anhydrase. Osmolyte counteraction of urea denaturation depends on osmolyte-protein pair.

Several phosphorylations are known to occur in the N-terminal transactivation domain of human p53. To explore the structural effects of these phosphorylations, we have chemically synthesized the unphosphorylated p53-(1-39) and its three phosphorylated analogs, phosphorylated at Ser-15, Thr-18, and Ser-20. p53-(1-39) and its Ser-15 and Thr-18 phosphorylated analogs were tested for interaction with p300. The order of binding affinities was similar to that derived from biochemical experiments with the whole protein, indicating functional integrity of the domain. Differences in chemical shifts and coupling constants indicate significant structural changes upon phosphorylations. The single tryptophan in the unphosphorylated domain has an emission maximum and a Stern-Volmer constant that are characteristics of tryptophans situated in protein interiors. The diffusion constant is monomer-like, with an axial ratio of 1:7.5, indicating a significant degree of compaction. Upon phosphorylations, the emission maximum and diffusion constant change significantly toward values that indicate more open conformations. Binding of the hydrophobic probe bis-1-anilino-8-naphthalenesulfonate to the unphosphorylated and one of the phosphorylated domains is also significantly different, suggesting different conformations. We propose that phosphorylations switch the largely folded transactivation domain to more open conformations that interact with transcription factors such as p300/cAMP- responsive element-binding protein-binding protein, leading to enhancement of gene expression.