Multivariate analysis of mean Raman spectra of erythrocytes for a fast analysis of the biochemical signature of ageing.

Ageing of red blood cells (RBC) is a physiological process, fundamental to ensure a proper blood homeostasis that, in vivo, balances the production of new cells and the removal of senescent erythrocytes. A detailed characterization at the cellular level of the progression of the ageing phenomenon can reveal biological, biophysical and biochemical fingerprints for diseases related to misbalances of the cell turnover and for blood pathologies. We applied Principal Components Analysis (PCA) to mean Raman spectra of single cells at different ageing times to rapidly highlight subtle spectral differences associated with conformational and biochemical modifications. Our results demonstrate a two-step ageing process characterized by a first phase in which proteins plays a relevant role, followed by a further cellular evolution driven by alterations in the membrane lipid contribution. Moreover, we used the same approach to directly analyse relevant spectral effects associated to reduction in Haemoglobin oxygenation level and membrane fluidity induced by the ageing. The method is robust and effective, allowing to classify easily the studied cells based on their age and morphology, and consequently to evaluate the biological quality of a blood sample.

A Rapid Unraveling of the Activity and Antibiotic Susceptibility of Mycobacteria.

The development of antibiotic-resistant bacteria is a worldwide health-related emergency that calls for new tools to study the bacterial metabolism and to obtain fast diagnoses. Indeed, the conventional analysis time scale is too long and affects our ability to fight infections. Slowly growing bacteria represent a bigger challenge, since their analysis may require up to months. Among these bacteria, Mycobacterium tuberculosis, the causative agent of tuberculosis, has caused more than 10 million new cases and 1.7 million deaths in 2016 only. We employed a particularly powerful nanomechanical oscillator, the nanomotion sensor, to characterize rapidly and in real time tuberculous and nontuberculous bacterial species, Mycobacterium bovis bacillus Calmette-Guérin and Mycobacterium abscessus, respectively, exposed to different antibiotics. Here, we show how high-speed and high-sensitivity detectors, the nanomotion sensors, can provide a rapid and reliable analysis of different mycobacterial species, obtaining qualitative and quantitative information on their responses to different drugs. This is the first application of the technique to tackle the urgent medical issue of mycobacterial infections, evaluating the dynamic response of bacteria to different antimicrobial families and the role of the replication rate in the resulting nanomotion pattern. In addition to a fast analysis, which could massively benefit patients and the overall health care system, we investigated the real-time responses of the bacteria to extract unique information on the bacterial mechanisms triggered in response to antibacterial pressure, with consequences both at the clinical level and at the microbiological level.

Insights into the morphological pattern of erythrocytes' aging: Coupling quantitative AFM data to microcalorimetry and Raman spectroscopy.

Erythrocytes (RBCs) constitute a very interesting class of cells both for their physiological function and for a variety of peculiarities. Due to their exceptionally strong relationship with the environment, the morphology and nanoscale characteristics of these cells can reveal their biochemical status and structural integrity. Among the possible subjects of investigations, the RBCs' ageing is of the utmost importance. This is a fundamental phenomenon that, in physiological conditions, triggers the cell turnover and ensures the blood homeostasis. With these premises, in recent years, we have presented an atomic force microscopy-based methodology to characterize the patterns of RBC ageing from the morphological point of view. In the present work, we used an ageing protocol more similar to the physiological conditions and we used differential scanning calorimetry and atomic force microscopy to probe the cross correlation between important structural and functional proteins. We also assessed the role played by fundamental structural and membrane proteins in the development of the most relevant morphological intermediates observed along the ageing. Furthermore, we coupled the morphological ageing patterns to the (bio)chemical alterations detected by Raman spectroscopy. This allowed identifying the chronology of the ageing morphologies and the metabolic pathways most involved in their development. As a whole, the present study provides the base to correlate specific molecular alterations to the development of structural anomalies, and these latter to the functional status of blood cells.

AFM nano-mechanical study of the beating profile of hiPSC-derived cardiomyocytes beating bodies WT and DM1.

Myotonic Dystrophy type 1 (DM1) is the most common form of muscular dystrophy in adults, characterized by a variety of multisystemic features and associated with cardiac anomalies. Among cardiac phenomena, conduction defects, ventricular arrhythmias, and dilated cardiomyopathy represent the main cause of sudden death in DM1 patients. Patient-specific induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a powerful in vitro model for molecular, biochemical, and physiological studies of disease in the target cells. Here, we used an Atomic Force Microscope (AFM) to measure the beating profiles of a large number of cells, organized in CM clusters (Beating Bodies, BBs), obtained from wild type (WT) and DM1 patients. We monitored the evolution over time of the frequency and intensity of the beating. We determined the variations between different BBs and over various areas of a single BB, caused by morphological and biomechanical variations. We exploited the AFM tip to apply a controlled force over the BBs, to carefully assess the biomechanical reaction of the different cell clusters over time, both in terms of beating frequency and intensity. Our measurements demonstrated differences between the WT and DM1 clusters highlighting, for the DM1 samples, an instability which was not observed in WT cells. We measured differences in the cellular response to the applied mechanical stimulus in terms of beating synchronicity over time and cell tenacity, which are in good agreement with the cellular behavior in vivo. Overall, the combination of hiPSC-CMs with AFM characterization can become a new tool to study the collective movements of cell clusters in different conditions and can be extended to the characterization of the BB response to chemical and pharmacological stimuli.

Erythrocyte's aging in microgravity highlights how environmental stimuli shape metabolism and morphology.

The determination of the function of cells in zero-gravity conditions is a subject of interest in many different research fields. Due to their metabolic unicity, the characterization of the behaviour of erythrocytes maintained in prolonged microgravity conditions is of particular importance. Here, we used a 3D-clinostat to assess the microgravity-induced modifications of the structure and function of these cells, by investigating how they translate these peculiar mechanical stimuli into modifications, with potential clinical interest, of the biochemical pathways and the aging processes. We compared the erythrocyte's structural parameters and selected metabolic indicators that are characteristic of the aging in microgravity and standard static incubation conditions. The results suggest that, at first, human erythrocytes react to external stimuli by adapting their metabolic patterns and the rate of consumption of the cell resources. On longer timeframes, the cells translate even small differences in the environment mechanical solicitations into structural and morphologic features, leading to distinctive morphological patterns of aging.

Nanotools and molecular techniques to rapidly identify and fight bacterial infections.

Reducing the emergence and spread of antibiotic-resistant bacteria is one of the major healthcare issues of our century. In addition to the increased mortality, infections caused by multi-resistant bacteria drastically enhance the healthcare costs, mainly because of the longer duration of illness and treatment. While in the last 20years, bacterial identification has been revolutionized by the introduction of new molecular techniques, the current phenotypic techniques to determine the susceptibilities of common Gram-positive and Gram-negative bacteria require at least two days from collection of clinical samples. Therefore, there is an urgent need for the development of new technologies to determine rapidly drug susceptibility in bacteria and to achieve faster diagnoses. These techniques would also lead to a better understanding of the mechanisms that lead to the insurgence of the resistance, greatly helping the quest for new antibacterial systems and drugs. In this review, we describe some of the tools most currently used in clinical and microbiological research to study bacteria and to address the challenge of infections. We discuss the most interesting advancements in the molecular susceptibility testing systems, with a particular focus on the many applications of the MALDI-TOF MS system. In the field of the phenotypic characterization protocols, we detail some of the most promising semi-automated commercial systems and we focus on some emerging developments in the field of nanomechanical sensors, which constitute a step towards the development of rapid and affordable point-of-care testing devices and techniques. While there is still no innovative technique that is capable of completely substituting for the conventional protocols and clinical practices, many exciting new experimental setups and tools could constitute the basis of the standard testing package of future microbiological tests.

Effect of electrical water bath stunning on physical reflexes of broilers: evaluation of stunning efficacy under field conditions.

The effects of different amounts and frequencies of stunning sine wave alternating current were investigated under field conditions. Seven hundred and fifty broilers were stunned in an electrical water bath with an average root mean square (RMS) current of 150, 200, and 250 mA and frequencies of 200, 400, 600, 800, and 1,200 Hz. The occurrence of corneal reflex, spontaneous eye blinking, and a positive response to a painful stimulus were monitored and recorded immediately after the stunning and at 20 s post-stun. Statistical analysis showed that the electrical stunning frequency (P=0.0004), the stunning RMS current (P<0.0001) and the interaction between stunning frequency and stunning current (P<0.0001) had a significant effect on the occurrence of animals experiencing an abolition of corneal reflex at 20 s post-stun.At a current of 150 mA, the probability of a successful stun was over 90% at 200 Hz, approximately 40% at 400 Hz, and below 5% for frequencies greater than 600 Hz. So, stunning at frequencies greater than 600 Hz cannot be recommended when a RMS current of 150 mA is applied. The maximum probability of a successful stun was obtained for a current level of 200 mA at 400 Hz and for a current level of 250 mA at 400 and 600 Hz, whereas the stunning treatments at 1,200 Hz provided the lowest probability of a successful stun. Assessment of spontaneous eye blinking and responses to comb pinching confirmed the indications coming from the analysis of corneal reflex.

Structural, morphological and nanomechanical characterisation of intermediate states in the ageing of erythrocytes.

The study of the mechanical properties of biosystems and the relationship with their biochemical and structural functionality is an increasingly interesting subject of investigation. In recent years, in particular, the use of the atomic force microscopy provides the tools for understanding the molecular basis of the mechanical behaviour of the biosystems. The ageing of erythrocytes [red blood cells (RBCs)] constitutes a particularly interesting subject of study because of its fundamental role in triggering the cell turnover by promoting the removal of malfunctioning RBCs when specific ageing markers appear on their surface. Moreover, it is also interesting to study the role that the variation in the cells mechanical properties plays in the progress of the phenomenon. In this study, the ageing of RBCs, accelerated by depleting the cells of their ATP, has been investigated by two methods. The first is a recently developed nondestructive approach that correlates the roughness of the plasma membrane to the mechanical characteristics and the structural integrity of the cell membrane-skeleton. The second consists in directly measuring the nanomechanical properties by acquiring and analysing force curves on the cell membrane. The application of the two methods allowed to define, for the first time, the general scheme of alterations the cells experience during the ageing. In particular, a progressive decrease of the membrane roughness, correlated to a weakening of the membrane-skeleton support, and a complex pattern of changes in the nanomechanical properties, which drives the morphological variation and the occurrence of the specific ageing markers on the cells, have been revealed.

AFM for diagnosis of nanocrystallization of steels in hardening processes.

INTRODUCTION: The aim of this study is to investigate the nanocrystallization of steels caused by the transformation from the austenitic to the martensitic phase induced by a severe plastic deformation (SPD) treatment. In this framework, we applied an air blast shot peening treatment, which is a simple protocol widely used for industrial purposes. METHODS: AISI 286 and AISI 316 specimens were peened for different times and polished using diamond pastes in order to remove corrugations higher than 1 mum. The characterization of the steel surfaces was performed by atomic force microscopy (AFM) operating in contact mode. Additional EDXD measurements were performed to confirm the phase transition. RESULTS AND DISCUSSION: An AFM-based characterization at nanometric level of the steel surfaces is provided. When the peening exceeds a threshold time that, as expected, depends on the steel composition, a uniform nanostructuration is detected. It is well known that such rearrangement is associated to the growth of a martensitic phase. To date, AFM has been employed in this field only for few applications and to solve specific problems. On the other hand, our results demonstrate that this is a useful technique for the characterization of hardened surfaces, especially when non-destructive sample preparation treatments are required. Moreover, we show that AFM can be a useful tool also for in situ industrial diagnostics of metallic parts.

Implementation of a bimorph-based aperture tapping-SNOM with an incubator to study the evolution of cultured living cells.

We present the implementation of a tapping-mode aperture scanning near-field optical microscope (Tapping-SNOM) to a Binder CB incubator (Istituto di Struttura della Materia, Rome, Italy). The microscope operates in the intermittent contact mode using a nonbent optical fibre allowing to reduce the perturbation exerted on the sample, while the incubator maintains a constant temperature, humidity and CO(2) level. This instrument can maintain and analyse in a controlled environment different samples, both organic and nonorganic. In particular, the Tapping-SNOM can study different cell lines at nanometric resolution and in physiological buffer, following the evolution of the living cells almost indefinitely. We will present several examples of the capabilities of the tapping scanning near-field optical microscope in the study of different lines of living cells, showing corresponding topographical, optical or phase-lag images of the live samples, evidencing the excellent stability, versatility and resolution of the system.

Roughness of the plasma membrane as an independent morphological parameter to study RBCs: a quantitative atomic force microscopy investigation.

A novel approach to the study of RBCs based on the collection of three-dimensional high-resolution AFM images and on the measure of the surface roughness of their plasma membrane is presented. The dependence of the roughness from several parameters of the imaging was investigated and a general rule for a trustful analysis and comparison has been suggested. The roughness of RBCs is a morphology-related parameter which has been shown to be characteristic of the single cells composing a sample, but independent of the overall geometric shape (discocyte or spherocyte) of the erythrocytes, thus providing extra-information with respect to a conventional morphology study. The use of the average roughness value as a label of a whole sample was tested on different kinds of samples. Analyzed data revealed that the quantitative roughness value does not change after treatment of RBCs with various commonly used fixation and staining methods while a drastic decrease occurs when studying cells with membrane-skeletal alteration both naturally occurring or artificially induced by chemical treatments. The present method provides a quantitative and powerful tool for a novel approach to the study of erythrocytes structure through an ultrastructural morphological analysis with the potential to give information, in a non-invasive way, on the RBCs function.

An AFM investigation of oligonucleotides anchored on unoxidized crystalline silicon surfaces.

Carboxylic terminated monolayers have been covalently attached on phosphorous doped crystalline (100) silicon surfaces using a cathodic electro grafting technique. The functionalization concentration and efficiency have been evaluated with different techniques. In particular, topographic images, performed with an atomic force microscope, were used to optimize the protocol in order to obtain a surface whose characteristics of uniformity and reproducibility are ideal for a bio-electronic device. Phase lag images of the functionalized surfaces were also performed, and show non-topographic structures that have been interpreted as areas of different molecule self-orientation. Poly-thymine oligonucleotides have been anchored on such a surface to form a nano-biosensing device capable to react selectively with a specific target molecule, a poly-adenine oligonucleotide. AFM images of high density (approximately 3x10(12) mol/cm2) single strand and double strand covered samples show toroidal shaped structures formed by the self-assembly of the oligonucleotides on the silicon surface.

AFM characterization of solid-supported lipid multilayers prepared by spin-coating.

Lipids are the principal components of biologically relevant structures as cellular membranes. They have been the subject of many studies due to their biological relevance and their potential applications. Different techniques, such as Langmuir-Blodgett and vesicle-fusion deposition, are available to deposit ordered lipid films on etched surfaces. Recently, a new technique of lipid film deposition has been proposed in which stacks of a small and well-controlled number of bilayers are prepared on a suitable substrate using a spin-coater. We studied the morphological properties of multi-layers made of cationic and neutral lipids (DOTAP and DOPC) and mixtures of them using dynamic mode atomic force microscopy (AFM). After adapting and optimizing, the spin-coating technique to deposit lipids on a chemically etched Silicon (1,0,0) substrate, a morphological nanometer-scale characterization of the aforementioned samples has been provided. The AFM study showed that an initial layer of ordered vesicles is formed and, afterward, depending on details of the spin-coating preparation protocol and to the dimension of the silicon substrate, vesicle fusion and structural rearrangements of the lipid layers may occur. The present data disclose the possibility to control the lipid's structures by acting on spin-coating parameters with promising perspectives for novel applications of lipid films.

Haem conformation of amphibian nytrosylhaemoglobins detected by XANES spectroscopy.

We investigated for the first time the haem stereochemistry in the nitrosylated derivative of two amphibian haemoglobins, Xenopus laevis and Ambystoma mexicanum, by means of X-ray absorption spectroscopy technique with the aim to explain the relationships between the active site structure and physiological function of these proteins, compared to that from humans. Our results show that while the Fe site local structure of human HbNO is modulated by an allosteric effector such as IHP shifting the T-R equilibrium towards the T-state, the Fe site local structure of amphibians HbNO is stabilized in a particularly tensed T-state also without IHP.

X-ray absorption near-edge spectroscopy of transferrins: a theoretical and experimental probe of the metal site local structure.

Proteins of the transferrin (Tf) family have a role in metal transport in vertebrates and have been extensively studied. The results here reported provide, for the first time, a detailed systematic comparison of metal sites in Tf complexes involving several atoms in the whole protein and in two different types of Tfs. The high interest in the structural variations induced in a metalloprotein upon the uptake of different metals is related to the hypothesis of the metals' involvement in some neuropathologies. We propose a comparative study of the X-ray absorption spectra at the K-edge of iron, copper, zinc and nickel in serotransferrin and ovotransferrin. The experimental data are simulated using an algorithm of the full multiple scattering method. Our results show that: (1) the local structure of each site (N-terminal and C-terminal) is correlated to the ligation state of the other site; (2) the difference between the two proteins is related to site local structure and depends on the metal ion nature being greater in the case of copper and zinc with respect to iron and nickel ions; (3) X-ray spectroscopy is confirmed as a suitable technique able to discriminate between coordination models proposed by X-ray diffraction.

Redox-induced structural dynamics of Fe-heme ligand in myoglobin by X-ray absorption spectroscopy.

The Fe(III) --> Fe(II) reduction of the heme iron in aquomet-myoglobin, induced by x-rays at cryogenics temperatures, produces a thermally trapped nonequilibrium state in which a water molecule is still bound to the iron. Water dissociates at T > 160 K, when the protein can relax toward its new equilibrium, deoxy form. Synchrotron radiation x-ray absorption spectroscopy provides information on both the redox state and the Fe-heme structure. Owing to the development of a novel method to analyze the low-energy region of x-ray absorption spectroscopy, we obtain structural pictures of this photo-inducible, irreversible process, with 0.02-0.06-A accuracy, on the protein in solution as well as in crystal. After photo-reduction, the iron-proximal histidine bond is shortened by 0.15 A, a reinforcement that should destabilize the iron in-plane position favoring water dissociation. Moreover, we are able to get the distance of the water molecule even after dissociation from the iron, with a 0.16-A statistical error.

Quantitative analysis of x-ray absorption near edge structure data by a full multiple scattering procedure: the Fe-CO geometry in photolyzed carbonmonoxy-myoglobin single crystal.

We report the first quantitative analysis of the Fe K-edge polarized x-ray absorption near edge structure of the iron protein carbonmonoxy-myoglobin (MbCO) single crystal and of its cryogenic photoproduct Mb(*)CO. The CO-Fe-heme local structure has been determined using a novel fitting procedure based on the full multiple scattering approach. The extracted local structure of Mb(*)CO includes a Fe-CO distance of (3.08+/-0.07) A, with a tilting angle between the heme normal and the Fe-C vector of (37+/-7) degrees, and a bending angle between the Fe-C vector and the C-O bond of (31+/-5) degrees.

Artificially induced unusual shape of erythrocytes: an atomic force microscopy study.

We used air operating atomic force microscopy (AFM) to study several morphological modifications of human erythrocytes, artificially produced by addition of exogenous agents including phospholipids, low ionic strength media and drugs. Most experiments were performed on unfixed samples to avoid treating red blood cells (RBCs) with chemical agents that can, in principle, induce morphological alteration. After detailed quantitative AFM characterization, the artificially produced abnormally shaped RBCs were compared with cells that occur with high incidence in blood pathologies. This morphological approach suggests a new strategy to describe and understand the biochemical and/or mechanical modifications responsible for the underlying pathologically induced changes and prove AFM to be a suitable tool to study erythrocyte deformation.

Structure of the Fe-heme in the hemodimeric hemoglobin from Scapharca inaequivalvis and in the T721 mutant: an X-ray absorption spectroscopic study at low temperature.

The Fe site structure in the recombinant wild-type and T721 mutant of the cooperative homodimeric hemoglobin (HbI) of the mollusc Scapharca itnaequivalvis has been investigated by measuring the Fe K-edge X-ray absorption near edge structure (XANES) spectra of their oxy, deoxy and carbonmonoxy derivatives, and the cryogenic photoproducts of the carbonmonoxy derivatives at T = 12 K. According to our results, the Fe site geometry in T72I HbI-CO is quite similar to that of human carbonmonoxy hemoglobin (HbA-CO), while in native HbI-CO it seems intermediate between that of HbA-CO and sperm whale MbCO. The XANES spectra of oxy and deoxy derivatives are similar to the homologous spectra of human HbA, except for T72I HbI, for which the absorption edge is blue-shifted (about + 1 eV) towards the spectrum of the oxy form. XANES spectra of the cryogenic photoproducts of HbA-CO (HbA*), HbI-CO (HbI*) and mutant HbI-CO (T72I HbI*) were acquired under continuous illumination at 12 K. The Fe-heme structures of the three photoproducts are similar; however, while in the case of HbA* and HbI* the data indicate incomplete structural relaxation of the Fe-heme towards its deoxy-like (T) form, the relaxation in T72I HbI* is almost completely towards the proposed "high affinity" Fe-heme structure of T72I HbI. This evidence suggests that minor tertiary restraints affect the Fe-heme dynamics of T72I HbI, corresponding to a reduction of the energy necessary for the T --> R structural transition, which can contribute to the observed dramatic enhancement in oxygen affinity of this hemoprotein, and the decreased cooperativity.

pH-dependent local structure of ferricytochrome c studied by x-ray absorption spectroscopy.

We have studied, using x-ray absorption spectroscopy by synchrotron radiation, the native state of the horse heart cytochrome c (N), the HCl denatured state (U(1) at pH 2), the NaOH denatured state (U(2) at pH 12), the intermediate HCl induced state (A(1) at pH 0.5), and the intermediate NaCl induced state (A(2) at pH 2). Although many results concerning the native and denatured states of this protein have been published, a site-specific structure analysis of the denatured and intermediate solvent induced states has never been attempted before. Model systems and myoglobin in different states of coordination are compared with cytochrome c spectra to have insight into the protein site structure in our experimental conditions. New features are evidenced by our results: 1) x-ray absorption near edge structure (XANES) of the HCl intermediate state (A(1)) presents typical structures of a pentacoordinate Fe(III) system, and 2) local site structures of the two intermediate states (A(1) and A(2)) are different.