Infrared spectromicroscopy of biochemistry in functional single cells

Over the years Fourier-Transform Infrared (FTIR) spectroscopy has been widely employed in the structural and functional characterization of biomolecules. The introduction of infrared (IR) microscopes and of synchrotron light sources has created expectations that FTIR could become a generally viable technique to study both structure and reactivity in vivo, inside single cells, by performing measurements that up to a few years ago were the preserve of in vitro experiments on purified macromolecules. In this review we present the state-of-the-art in the application of FTIR spectromicroscopy as a technique for the study of structure and dynamics in single cells, we discuss the performance requirements for this application and review developments in sample handling methods.     

Nanoparticle microinjection and Raman spectroscopy as tools for nanotoxicology studies

Microinjection techniques and Raman spectroscopy have been combined to provide a new methodology to investigate the cytotoxic effects due to the interaction of nanomaterials with cells. In the present work, this novel technique has been used to investigate the effects of Ag and Fe(3)O(4) nanoparticles on Hela cells. The nanoparticles are microinjected inside the cells and these latter ones are probed by means of Raman spectroscopy after a short incubation time, in order to highlight the first and impulsive mechanisms developed by the cells to counteract the presence of the nanoparticles. The results put in evidence a different behaviour of the cells treated with nanoparticles in comparison with the control cells; these differences are supposed to be generated by an emerging oxidative stress due to the nanoparticles. The achieved results demonstrate the suitability of the proposed method as a new tool for nanotoxicity studies.     

Mirror‐Image Organometallic Osmium Arene Iminopyridine Halido Complexes Exhibit Similar Potent Anticancer Activity

Four chiral Os^II arene anticancer complexes have been isolated by fractional crystallization. The two iodido complexes, (S_Os,S_C)‐[Os(η⁶‐p‐cym)(ImpyMe)I]PF₆ (complex 2 , (S)‐ImpyMe: N‐(2‐pyridylmethylene)‐(S)‐1‐phenylethylamine) and (R_Os,R_C)‐[Os(η⁶‐p‐cym)(ImpyMe)I]PF₆ (complex 4 , (R)‐ImpyMe: N‐(2‐pyridylmethylene)‐(R)‐1‐phenylethylamine), showed higher anticancer activity (lower IC₅₀ values) towards A2780 human ovarian cancer cells than cisplatin and were more active than the two chlorido derivatives, (S_Os,S_C)‐[Os(η⁶‐p‐cym)(ImpyMe)Cl]PF₆, 1 , and (R_Os,R_C)‐[Os(η⁶‐p‐cym)(ImpyMe)Cl]PF₆, 3 . The two iodido complexes were evaluated in the National Cancer Institute 60‐cell‐line screen, by using the COMPARE algorithm. This showed that the two potent iodido complexes, 2 (NSC: D‐758116/1) and 4 (NSC: D‐758118/1), share surprisingly similar cancer cell selectivity patterns with the anti‐microtubule drug, vinblastine sulfate. However, no direct effect on tubulin polymerization was found for 2 and 4 , an observation that appears to indicate a novel mechanism of action. In addition, complexes 2 and 4 demonstrated potential as transfer‐hydrogenation catalysts for imine reduction.     

Multi‐faceted Reactivity of Alkyltellurophenols Towards Peroxyl Radicals: Catalytic Antioxidant Versus Thiol‐Depletion Effect

Hydroxyaryl alkyl tellurides are effective antioxidants both in organic solution and aqueous biphasic systems. They react by an unconventional mechanism with ROO^. radicals with rate constants as high as 10⁷ M ^?1 s^?1 at 303 K, outperforming common phenols. The reactions proceed by oxygen atom transfer to tellurium followed by hydrogen atom transfer to the resulting RO^. radical from the phenolic OH. The reaction rates do not reflect the electronic properties of the ring substituents and, because the reactions occur in a solvent cage, quenching is more efficient when the OH and TeR groups have an ortho arrangement. In the presence of thiols, hydroxyaryl alkyl tellurides act as catalytic antioxidants towards both hydroperoxides (mimicking the glutathione peroxidases) and peroxyl radicals. The high efficiency of the quenching of the peroxyl radicals and hydroperoxides could be advantageous under normal cellular conditions, but pro‐oxidative (thiol depletion) when thiol concentrations are low.     

Fluorine Substitution Effects on Flexibility and Tunneling Pathways: The Rotational Spectrum of 2‐Fluorobenzylamine

The effect of ring fluorination on the structural and dynamical properties of the flexible model molecule 2‐fluorobenzylamine has been studied by rotational spectroscopy in free‐jet expansion and quantum chemical methods. The complete potential energy surface originating from the flexibility of the aminic side chain has been calculated at the B3LYP/6‐311++G** level of theory and the stable geometries were also characterized with MP2/6‐311++G**. The rotational spectra show the presence of two of the predicted four stable conformers: the global minimum (I), in which the side chain’s dihedral angle with the phenyl plane is almost perpendicular, is stabilized by an intramolecular hydrogen bond between the fluorine atom and one hydrogen of the aminic group; and a second conformer II (E_II?E_I≈5 kJ mol^?1) in which the dihedral angle is smaller and the amino group points towards the aromatic ortho hydrogen atom. This conformation is characterized by a tunneling motion between two equivalent positions of the amino group with respect to the phenyl plane, which splits the rotational transition. The ortho fluorination increases, with respect to benzylamine, the tunneling splitting of this motion by four orders of magnitude. The motion is analyzed with a one‐dimensional flexible model, which allows estimation of the energy barrier for the transition state as approximately 8.0 kJ mol^?1.     

Dual-color bioluminescent bioreporter for forensic analysis: evidence of androgenic and anti-androgenic activity of illicit drugs

Bioassays represent promising complementary techniques to conventional analytical approaches used in doping analysis to detect illicit drugs like anabolic-androgenic steroids (AAS). The fact that all AAS share a common mechanism of action via the human androgen receptor (hAR) enables the use of bioassays, relying on the activation of hAR as antidoping screening tools. Previously, we developed a dual-color bioreporter based on yeast cells engineered to express hAR and androgen response elements driving the expression of the bioluminescent (BL) reporter protein Photinus pyralis luciferase. A second reporter protein, the red-emitting luciferase PpyRE8, was introduced in the bioreporter as internal viability control. Here, we report the first forensic application of a straightforward, accurate, and cost-effective bioassay, relying on spectral resolution of the two BL signals, in 96-microwell format. The bioreporter responds to dihydrotestosterone as reference androgen in a concentration-dependent manner from 0.08 to 1,000 nM with intra- and inter-assay variation coefficients of 11.4 % and 13.1 %, respectively. We also demonstrated the suitability of this dual-color bioreporter to assess (anti)-androgenic activity of pure AAS, mixtures of AAS, and other illicit drugs provided by the Scientific Police. Significant anti-androgenic activity was observed in samples labeled as marijuana and hashish, containing Δ⁹-tetrahydrocannabinol as major constituent.

A New Alkaliphilic Cold-Active Esterase from the Psychrophilic Marine Bacterium Rhodococcus sp.: Functional and Structural Studies and Biotechnological Potential

The special features of cold-adapted lipolytic biocatalysts have made their use possible in several industrial applications. In fact, cold-active enzymes are known to be able to catalyze reactions at low temperatures, avoiding side reactions taking place at higher temperatures and preserving the integrity of products. A lipolytic gene was isolated from the Arctic marine bacterium Rhodococcus sp. AW25M09 and expressed in Escherichia coli as inclusion bodies. The recombinant enzyme (hereafter called RhLip) showed interesting cold-active esterase activity. The refolded purified enzyme displayed optimal activity at 30 °C and was cold-active with retention of 50 % activity at 10 °C. It is worth noting that the optimal pH was 11, and the low relative activity below pH 10 revealed that RhLip was an alkaliphilic esterase. The enzyme was active toward short-chain p-nitrophenyl esters (C2–C6), displaying optimal activity with the butyrate (C4) ester. In addition, the enzyme revealed a good organic solvent and salt tolerance. These features make this an interesting enzyme for exploitation in some industrial applications.     

Almost periodic invariant tori for the NLS on the circle

In this paper we study the existence and linear stability of almost periodic solutions for a NLS equation on the circle with external parameters. Starting from the seminal result of Bourgain in [15] on the quintic NLS, we propose a novel approach allowing to prove in a unified framework the persistence of finite and infinite dimensional invariant tori, which are the support of the desired solutions. The persistence result is given through a rather abstract “counter-term theorem” à la Herman, directly in the original elliptic variables without passing to action-angle ones. Our framework allows us to find “many more” almost periodic solutions with respect to the existing literature and consider also non-translation invariant PDEs.     

Exchange bias in nanopatterned Co antidots prepared by self-assembling polystyrene nanospheres

The exchange bias properties of nanopatterned thin films of Co, on top of which a native Co-oxide layer develops spontaneously, are studied by means of magnetic and magneto-resistance measurements. Both continuous and patterned films are investigated, the latter in the form of antidot arrays prepared with the self-assembling polystyrene nanospheres technique. The obtained antidot arrays are in the hexagonal close-packed configuration and cover a surface area of several square millimetres. Nanopatterned samples turn out to have a very good repeatability of their magnetic and magneto-resistive properties. The presence of a native oxide is responsible for the development of an exchange bias effect at temperatures below ~150 K, which has been reported both on hysteresis loops and on magneto-resistance curves; these consist of a superposition of an anisotropic magneto resistance (AMR) effect and a giant magneto-resistance (GMR)-like effect. The determination of the bias field by means of the two different sets of data is consistent and gives a complete picture of the phenomenology in this kind of nanopatterned magnetic systems.     

In Vitro Assessment of Tobacco Smoke Toxicity at the BBB: Do Antioxidant Supplements Have a Protective Role?

Background

Calcitonin gene-related peptide (CGRP) has a key role in migraine pathophysiology and is associated with activation of the trigeminovascular system. The trigeminal ganglion, storing CGRP and its receptor components, projects peripheral to the intracranial vasculature and central to regions in the brainstem with Aδ- and C-fibers; this constitutes an essential part of the pain pathways activated in migraine attacks. Therefore it is of importance to identify the regions within the brainstem that processes nociceptive information from the trigeminovascular system, such as the spinal trigeminal nucleus (STN) and the C1-level of the spinal cord. Immunohistochemistry was used to study the distribution and relation between CGRP and its receptor components - calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1) - in human and rat STN and at the C1-level, using a set of newly well characterized antibodies. In addition, double-stainings with CGRP and myelin basic protein (MBP, myelin), synaptophysin (synaptic vesicles) or IB4 (C-fibers in general) were performed.

Results

In the STN, the highest density of CGRP immunoreactive fibers were found in a network around fiber bundles in the superficial laminae. CLR and RAMP1 expression were predominately found in fibers in the spinal trigeminal tract region, with some fibers spanning into the superficial laminae. Co-localization between CGRP and its receptor components was not noted. In C1, CGRP was expressed in fibers of laminae I and II. The CGRP staining was similar in rat, except for CGRP positive neurons that were found close to the central canal. In C1, the receptor components were detected in laminae I and II, however these fibers were distinct from fibers expressing CGRP as verified by confocal microscopy.

Conclusions

This study demonstrates the detailed expression of CGRP and its receptor components within STN in the brainstem and in the spinal cord at C1-level, and shows the possibility of CGRP acting postjunctionally in these areas putatively involved in primary headaches.