Atomic Force Microscopy Applied to the Study of Tauopathies.

Atomic force microscopy (AFM) is a scanning probe microscopy technique which has a physical principle, the measurement of interatomic forces between a very thin tip and the surface of a sample, allowing the obtaining of quantitative data at the nanoscale, contributing to the surface study and mechanical characterization. Due to its great versatility, AFM has been used to investigate the structural and nanomechanical properties of several inorganic and biological materials, including neurons affected by tauopathies. Tauopathies are neurodegenerative diseases featured by aggregation of phosphorylated tau protein inside neurons, leading to functional loss and progressive neurotoxicity. In the broad universe of neurodegenerative diseases, tauopathies comprise the most prevalent, with Alzheimer's disease as its main representative. This review highlights the use of AFM as a suitable research technique for the study of cellular damages in tauopathies, even in early stages, allowing elucidation of pathogenic mechanisms of these diseases.

Nanoscale Dynamics of Streptococcal Adhesion to AGE-Modified Collagen.

The adhesion of initial colonizers such as Streptococcus mutans to collagen is critical for dentinal and root caries progression. One of the most described pathological and aging-associated changes in collagen-including dentinal collagen-is the generation of advanced glycation end-products (AGEs) such as methylglyoxal (MGO)-derived AGEs. Despite previous reports suggesting that AGEs alter bacterial adhesion to collagen, the biophysics driving oral streptococcal attachment to MGO-modified collagen remains largely understudied. Thus, the aim of this work was to unravel the dynamics of the initial adhesion of S. mutans to type I collagen in the presence and absence of MGO-derived AGEs by employing bacterial cell force spectroscopy with atomic force microscopy (AFM). Type I collagen gels were treated with 10 mM MGO to induce AGE formation, which was characterized with microscopy and enzyme-linked immunosorbent assay. Subsequently, AFM cantilevers were functionalized with living S. mutans UA 159 or Streptococcus sanguinis SK 36 cells and probed against collagen surfaces to obtain force curves displaying bacterial attachment in real time, from which the adhesion force, number of events, Poisson analysis, and contour and rupture lengths for each individual detachment event were computed. Furthermore, in silico computer simulation docking studies between the relevant S. mutans UA 159 collagen-binding protein SpaP and collagen were computed, in the presence and absence of MGO. Overall, results showed that MGO modification increased both the number and adhesion force of single-unbinding events between S. mutans and collagen, without altering the contour or rupture lengths. Both experimental and in silico simulations suggest that this effect is due to increased specific and nonspecific forces and interactions between S. mutans UA 159 and MGO-modified collagen substrates. In summary, these results suggest that collagen alterations due to aging and glycation may play a role in early bacterial adherence to oral tissues, associated with conditions such as aging or chronic hyperglycemia, among others.

Role of galectin-3 in the elastic response of radial growth phase melanoma cancer cells.

Melanoma is originated from the malignant transformation of the melanocytes and is characterized by a high rate of invasion, the more serious stage compromising deeper layers of the skin and eventually leading to the metastasis. A high mortality due to melanoma lesion persists because most of melanoma lesions are detected in advanced stages, which decreases the chances of survival. The identification of the principal mechanics implicated in the development and progression of melanoma is essential to devise new early diagnosis strategies. Cell mechanics is related with a lot of cellular functions and processes, for instance motility, differentiation, migration and invasion. In particular, the elastic modulus (Young's modulus) is a very explored parameter to describe the cell mechanical properties; most cancer cells reported in the literature smaller elasticity modulus. In this work, we show that the elastic modulus of melanoma cells lacking galectin-3 is significantly lower than those of melanoma cells expressing galectin-3. More interestingly, the gradient of elastic modulus in cells from the nuclear region towards the cell periphery is more pronounced in shGal3 cells. RESEARCH HIGHLIGHTS: AFM imaging and force spectroscopy were used to investigate the morphology and elasticity properties of healthy HaCaT cells and melanoma cells WM1366, with (shSCR) and without (shGal3) expression of galectin-3. It is shown the effect of galectin-3 protein on the elastic properties of cells: the cells without expression of galectin-3 presents lower elastic modulus. By the results, we suggest here that galectin-3 could be used as an effective biomarker of malignancy in both melanoma diagnostic and prognosis.

Methodologies and models for measuring viscoelastic properties of cancer cells: Towards a universal classification.

Different methods and several physical models exist to study cell viscoelasticity with the atomic force microscope (AFM). In search of a robust mechanical classification of cells through AFM, in this work, viscoelastic parameters of the cancer cell lines MDA-MB-231, DU-145, and MG-63 are obtained using two methodologies; through force-distance and force-relaxation curves. Four mechanical models were applied to fit the curves. The results show that both methodologies agree qualitatively on the parameters that quantify elasticity but disagree on the parameters that account for energy dissipation. The Fractional Zener (FZ) model represents well the information given by the Solid Linear Standard and Generalized Maxwell models. The Fractional Kelvin (FK) model concentrates the viscoelastic information mainly in two parameters, which could be an advantage over the other models. Therefore, the FZ and FK models are proposed as the basis for the classification of cancer cells. However, more research using these models is needed to obtain a broader view of the meaning of each parameter and to be able to establish a relationship between the parameters and the cellular components.

Differences in erythrocytes size and shape in prediabetes and diabetes assessed by two microscopy techniques and its association with dietary patterns. Pilot study.

Hemorheology and microcirculation alterations are caused by erythrocyte size and shape (ESS) modifications. People´s diets can alter erythrocyte functions and membrane fluidity by changing cell membrane components. The aim was to identify differences in ESS obtained by scanning electron (SEM) and atomic force microscopy (AFM) in people with prediabetes and type 2 diabetes (T2DM) and assess their relationship with dietary patterns. The study population included 31 participants (14 healthy, 11 with prediabetes, and 6 with T2DM). Dietary intake was assessed by a food frequency questionnaire, and dietary patterns were obtained using principal component analysis. ESS (diameter, height, axial ratio, thickness, and concave depth) were obtained by SEM and AFM. Differences in ESS between groups were observed with SEM (height) and AFM (height, axial ratio, and concave depth). T2DM presented smaller erythrocytes, more elongated and more altered forms. Two dietary patterns were identified: (1) Unhealthy: more refined cereals, high-fat dairy, fast food, sugary beverages, and fewer fruits, fish, seafood, low-fat dairy, and water. (2) Prudent: higher consumption of refined cereals, vegetables, poultry, low-fat dairy and nuts, and lower tortillas, eggs, high-fat dairy, and legumes. Tertile 3 of the Unhealthy dietary pattern had 80% of healthy participants. A difference in diameter and height (0.44 and 0.32 µm, respectively) obtained by SEM was observed when comparing tertile 2 (smaller erythrocytes) versus tertile 3 in the Unhealthy dietary pattern. SEM and AFM are excellent tools to assess ESS. Unhealthy dietary patterns might be associated with altered ESS. HIGHLIGHTS: SEM and AFM are excellent tools to assess erythrocyte size and shape modifications. Two dietary patterns were identified: healthy and prudent. Smaller erythrocytes were observed in the second tertile of the unhealthy pattern.

Ultrastructural characterisation of young and aged dental enamel by atomic force microscopy.

Recent advances in atomic force microscopy (AFM) have allowed the characterisation of dental-associated biomaterials and biological surfaces with high resolution. In this context, the topography of dental enamel - the hardest mineralised tissue in the body - has been explored with AFM-based approaches at the microscale. With age, teeth are known to suffer changes that can impact their structural stability and function; however, changes in enamel structure because of ageing have not yet been explored with nanoscale resolution. Therefore, the aim of this exploratory work was to optimise an approach to characterise the ultrastructure of dental enamel and determine potential differences in topography, hydroxyapatite (HA) crystal size, and surface roughness at the nanoscale associated to ageing. For this, a total of six teeth were collected from human donors from which enamel specimens were prepared. By employing intermittent contact (AC mode) imaging, HA crystals were characterised in both transversal and longitudinal orientation (respect to surface plane) with high resolution in environmental conditions. The external enamel surface displayed the presence of a pellicle-like coating on its surface that was not observable on cleaned specimens. Acid-etching exposed crystals that were imaged and morphologically characterised in high resolution at the nanoscale in both the external and internal regions of enamel in older and younger specimens. Our results demonstrated important individual variations in HA crystal width and roughness parameters across the analysed specimens; however, an increase in surface roughness and decrease in HA width was observed for the pooled older external enamel group compared to younger specimens. Overall, high-resolution AFM was an effective approach for the qualitative and quantitative characterisation of human dental enamel ultrastructure. Future work should focus on exploring the ageing of dental enamel with increased sample sizes to compensate for individual differences as well as other potential confounding factors such as behavioural habits and mechanical forces.

Dry Two-Step Self-Assembly of Stable Supported Lipid Bilayers on Silicon Substrates.

Artificial membranes are models for biological systems and are important for applications. We introduce a dry two-step self-assembly method consisting of the high-vacuum evaporation of phospholipid molecules over silicon, followed by a subsequent annealing step in air. We evaporate dipalmitoylphosphatidylcholine (DPPC) molecules over bare silicon without the use of polymer cushions or solvents. High-resolution ellipsometry and AFM temperature-dependent measurements are performed in air to detect the characteristic phase transitions of DPPC bilayers. Complementary AFM force-spectroscopy breakthrough events are induced to detect single- and multi-bilayer formation. These combined experimental methods confirm the formation of stable non-hydrated supported lipid bilayers with phase transitions gel to ripple at 311.5 ± 0.9 K, ripple to liquid crystalline at 323.8 ± 2.5 K and liquid crystalline to fluid disordered at 330.4 ± 0.9 K, consistent with such structures reported in wet environments. We find that the AFM tip induces a restructuring or intercalation of the bilayer that is strongly related to the applied tip-force. These dry supported lipid bilayers show long-term stability. These findings are relevant for the development of functional biointerfaces, specifically for fabrication of biosensors and membrane protein platforms. The observed stability is relevant in the context of lifetimes of systems protected by bilayers in dry environments.

Advanced Microtexture Study of Anacardium occidentale L. Leaf Surface From the Amazon by Fractal Theory.

This work applies stereometric parameters and fractal theory to characterize the structural complexity of the 3D surface roughness of Anacardium occidentale L. leaf using atomic force microscopy (AFM) measurements. Surface roughness was studied by AFM in tapping mode, in air, on square areas of 6,400 and 10,000 µm2. The stereometric analyses using MountainsMap Premium and WSXM software provided detailed information on the 3D surface topography of the samples. These data showed that the morphology of the abaxial and adaxial side of the cashew leaf is different, which was also observed in relation to their microtextures. Fractal analysis showed that the adaxial and abaxial sides have strong microtexture homogeneity, but the adaxial side presented higher surface entropy. These results show that image processing associated with fractal theory can be an indispensable tool for identifying plant species by their leaves because this species has singularities on each side of the leaf.

Advanced micromorphology study of microbial films grown on Kefir loaded with Açaí extract.

In this work, an advanced analysis of the 3D surface microtexture of the microbial films grown on Kefir loaded with Açaí extract was performed. Atomic force microscopy was used to characterize the 3D surface microtexture data in correlation with the stereometric analyses to allow a better understanding of the surface micromorphology consistent with ISO 25178-2: 2012. Two new parameters, fractal succolarity and fractal lacunarity, have been inserted for a quantitative approach to microtexture. The results revealed that the morphology was affected by the increase of the Açaí concentration in biofilms, as well as the fractal succolarity and lacunarity. Adhesive bacteria of the genus Lactobacillus were observed for the lowest concentrations of Açaí. Moreover, it was found that the surface of the biofilms has shown saturation when the concentration has changed from 4 to 6 % of Açaí. These results are of great interest in the characterization of surfaces with promising application like skin dressing.

Synthesis and Characterization of Nanostructured Lipid Nanocarriers for Enhanced Sun Protection Factor of Octyl p-methoxycinnamate.

Sunlight is important to health, but higher exposure to radiation causes early aging of the skin and skin damage that can lead to skin cancers. This study aimed at producing a stable octyl p-methoxycinnamate (OMC)-loaded nanostructured lipid carrier (NLC) sunscreen, which can help in the photoprotective effect. NLC was produced by emulsification-sonication method and these systems were composed of myristyl myristate (MM), caprylic capric triglyceride (CCT), Tween® 80 (TW), and soybean phosphatidylcholine (SP) and characterized by dynamic light scattering (DLS), zeta potential (ZP) measurement, atomic force microscopy (AFM), scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and in vitro release studies. Pre-formulation studies were performed changing TW concentrations and no differences were found at concentrations of 1.0 and 2.0%. Two selected formulations were designed and showed an average size of 91.5-131.7, polydispersity index > 0.2, and a negative value of ZP. AFM presented a sphere-like morphology and SEM showed ability to form a thin film. DSC exhibited that the incorporation of OMC promoted reduction of enthalpy due to formation of a more amorphous structure. Drug release shows up to 55.74% and 30.57%, and this difference could be related to the presence of SP in this formulation that promoted a more amorphous structure; the release mechanism study indicated Fickian diffusion and relaxation. Sun protection factor (SPF) evaluation was performed using NLC and presented values around 40, considerably higher than those observed in the literature. The developed formulations provide a beneficial alternative to conventional sunscreen formulations.

Interfering with the Folding of Group A Streptococcal pili Proteins.

Gram-positive bacteria use their adhesive pili to attach to host cells during early stages of a bacterial infection. These extracellular hair-like appendages experience mechanical stresses of hundreds of picoNewtons; however, the presence of an internal isopeptide bond prevents the pilus protein from unfolding. Here, we describe a method to interfere with nascent pili proteins through a peptide that mimics one of the ß-strands of the molecule. By using AFM-based force spectroscopy, we study the isopeptide bond formation and the effect of the peptide in the elasticity of the pilus protein. This method could be used to afford a new strategy for mechanically targeted antibiotics by simply blocking the folding of the bacterial pilus protein.

Sensing molecular organizational changes through the catalytic activity of acetylcholinesterase from erythrocyte membranes in Langmuir-Blodgett films.

Langmuir films prepared from bovine erythrocyte membranes (LFBEM) were studied and transferred to alkylated glasses (Langmuir-Blodgett films, LBBEM) in order to assess the effects of membrane molecular packing on Bovine Erythrocyte Acetylcholinesterase (BEA) catalytic activity. Surface pressure (π) vs Area isotherms showed three 2D-transitions at ~7, ~18 and ~44 mN/m and a collapse pressure at πc = 49 mN/m. The 0-12-0 mN/m compression-decompression cycles resulted reversible while those 0-40-0 mN/m exhibited a significant hysteresis. Taken together, EFM, BAM and AFM images and the stability of the film after 3C-D cycles, we can suggest that over the air-water interface as well as over the silanized glass substrate the surface is mostly covered by a monolayer with a few particles dispersed. Acetylthiocholine hydrolysis was assayed with BEA in bovine erythrocyte membrane suspensions (SBEM) and in LBBEM packed at 10 (LBBEM,10) and 35 mN/m (LBBEM,35), which gave the following kinetic parameters: Vmax = 3.41 ± 0.15, 0.021 ± 0.002 and 0.030 ± 0.003 nmol.min-1·µg prot-1 and KM = 0.11 ± 0.02, 0.047 ± 0.017 and 0.026 ± 0.017 mM, respectively. Although from SBEM to LBBEM we lost active enzyme, the catalytic efficiency (Vmax/KM) increased ~750 times. Eugenol and 1,8-cineol inhibited BEA catalytic activity in LBBEM,35. Our results demonstrate the transmission of information between the membrane and the environment within the subphase immediately below the membrane, where anchored proteins are hosted. This was reflected by the membrane packing-induced modulation of BEA catalytic activity. Furthermore, LBBEM provides a proof of concept for the development of biosensors to screen new green pesticides acting through BEA interaction.

Viscoelastic properties of doxorubicin-treated HT-29 cancer cells by atomic force microscopy: the fractional Zener model as an optimal viscoelastic model for cells.

The malignancy of cancer cells and their response to drug treatments have been traditionally studied using solely their elastic properties. However, the study of the combined viscous and elastic properties provides a richer description of the mechanics of the cell, and achieves a more precise assessment of the effect exerted by anti-cancer treatments. We used an atomic force microscope to obtain the morphological, elastic and viscous properties of HT-29 colorectal cancer cells. Changes in these parameters were observed during exposure of the cells to doxorubicin at different times. The elastic properties were analyzed using the Hertz and Sneddon models. Furthermore, we analyzed the data to study the viscoelasticity of the cells comparing the models known as the standard linear solid, fractional Zener, generalized Maxwell, and power law. A discussion about the optimal model based in the accuracy and physical assumptions for this particular system is included. From the morphological data and viscoelasticity of HT-29 cells exposed to doxorubicin, we found that some parameters were affected differently at shorter or longer exposure times. For instance, the relaxation time suggests a measure of the cell to self-heal and it was observed to increase at shorter exposure times and then to reduce for longer exposure times to the drug. The fractional Zener model better described the mechanical properties of the cell due to the reduced number of parameters and the quality of the fit to experimental data.

A highly specific and sensitive nanoimmunosensor for the diagnosis of neuromyelitis optica spectrum disorders.

A precise diagnosis for neuromyelitis optica spectrum disorders (NMOSD) is crucial to improve patients' prognostic, which requires highly specific and sensitive tests. The cell-based assay with a sensitivity of 76% and specificity of 100% is the most recommended test to detect anti-aquaporin-4 antibodies (AQP4-Ab). Here, we tested four AQP4 external loop peptides (AQP461-70, AQP4131-140, AQP4141-150, and AQP4201-210) with an atomic force microscopy nanoimmunosensor to develop a diagnostic assay. We obtained the highest reactivity with AQP461-70-nanoimunosensor. This assay was effective in detecting AQP4-Ab in sera of NMOSD patients with 100% specificity (95% CI 63.06-100), determined by the cut-off adhesion force value of 241.3 pN. NMOSD patients were successfully discriminated from a set of healthy volunteers, patients with multiple sclerosis, and AQP4-Ab-negative patients. AQP461-70 sensitivity was 81.25% (95% CI 56.50-99.43), slightly higher than with the CBA method. The results with the AQP461-70-nanoimmunosensor indicate that the differences between NMOSD seropositive and seronegative phenotypes are related to disease-specific epitopes. The absence of AQP4-Ab in sera of NMOSD AQP4-Ab-negative patients may be interpreted by assuming the existence of another potential AQP4 peptide sequence or non-AQP4 antigens as the antibody target.

Evaluation of Nanomechanical Properties of Tomato Root by Atomic Force Microscopy.

Here, different tissue surfaces of tomato root were characterized employing atomic force microscopy on day 7 and day 21 of growth through Young's modulus and plasticity index. These parameters provide quantitative information regarding the mechanical behavior of the tomato root under fresh conditions in different locations of the cross-section of root [cell surface of the epidermis, parenchyma (Pa), and vascular bundles (Vb)]. The results show that the mechanical parameters depend on the indented region, tissue type, and growth time. Thereby, the stiffness increases in the cell surface of epidermal tissue with increasing growth time (from 9.19 ± 0.68 to 13.90 ± 1.68 MPa) and the cell surface of Pa tissue displays the opposite behavior (from 1.74 ± 0.49 to 0.48 ± 0.55); the stiffness of cell surfaces of Vb tissue changes from 10.60 ± 0.58 to 6.37 ± 0.53 MPa, all cases showed a statistical difference (p < 0.05). Viscoelastic behavior dominates the mechanical forces in the tomato root. The current study is a contribution to a better understanding of the cell mechanics behavior of different tomato root tissues during growth.

Alterations to plasma membrane lipid contents affect the biophysical properties of erythrocytes from individuals with hypertension.

Genetic and environmental factors may contribute to high blood pressure, which is termed essential hypertension. Hypertension is a major independent risk factor for cardiovascular disease, stroke and renal failure; thus, elucidation of the etiopathology of hypertension merits further research. We recently reported that the platelets and neutrophils of patients with hypertension exhibit altered biophysical characteristics. In the present study, we assessed whether the major structural elements of erythrocyte plasma membranes are altered in individuals with hypertension. We compared the phospholipid (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingosine) and cholesterol contents of erythrocytes from individuals with hypertension (HTN) and healthy individuals (HI) using LC/MS-MS. HTN erythrocytes contained higher phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine contents and a lower cholesterol content than HI erythrocytes. Furthermore, atomic force microscopy revealed important morphological changes in HTN erythrocytes, which reflected the increased membrane fragility and fluidity and higher levels of oxidative stress observed in HTN erythrocytes using spectrophotofluorometry, flow cytometry and spectrometry. This study reveals that alterations to the lipid contents of erythrocyte plasma membranes occur in hypertension, and these alterations in lipid composition result in morphological and physiological abnormalities that modify the dynamic properties of erythrocytes and contribute to the pathophysiology of hypertension.

The effect of nanoscale surface electrical properties of partially biodegradable PEDOT-co-PDLLA conducting polymers on protein adhesion investigated by atomic force microscopy.

This study used atomic force microscopy (AFM) to elucidate the interaction of fibronectin (FN) on a conducting and partially biodegradable copolymer of poly(3,4-ethylenedioxythiophene) and poly(d,l-lactic acid) (PEDOT-co-PDLLA) in three different proportions (1:05, 1:25 and 1:50). The copolymers with higher PEDOT:PDLLA content ratios (1:05 and 1:25) had higher surface roughness, water contact angle, with current and conductivity occurring at discrete large grain structures on the surface. In contrast, the lower PEDOT:PDLLA content ratio (1:50) did not show high conductivity grains but showed homogenous surface conductivity across the entire surface. Using FN-functionalized AFM probes, force measurements showed that the copolymers with higher PEDOT content (1:05 and 1:25) had significantly lower adhesion forces (~0.2-0.3 nN), while the copolymer with the lower content of PEDOT (1:50) had stronger FN interactions with significantly higher adhesion forces of 1 nN. By correlating the spatially distributed electrical surfaces with FN interactions, we observed that the synthesis of 1:50 PEDOT:PDLLA produced more uniformly doped polymer films that facilitated FN adsorption through favorable interactions with accessible sulfate dopants. Importantly, these findings are correlated with previous studies showing increased stem cell migration and differentiation on 1:50 PEDOT:PDLLA surfaces compared with surfaces with 1:05 and 1:25 PEDOT:PDLLA ratios.

Antibody-mediated biorecognition of myelin oligodendrocyte glycoprotein: computational evidence of demyelination-related epitopes.

Antigen-antibody interaction is crucial in autoimmune disease pathogenesis, as multiple sclerosis and neuromyelitis optica. Given that, autoantibodies are essential biomolecules, of which the myelin oligodendrocyte glycoprotein (MOG) can figure as a target. Here we combined Molecular Dynamics (MD), Steered Molecular Dynamics (SMD), and Atomic Force Microscope (AFM) to detail MOG recognition by its specific antibody. The complex model consisted of the MOG external domain interacting with an experimental anti-MOG antibody from the Protein Data Bank (1PKQ). Computational data demonstrated thirteen MOG residues with a robust contribution to the antigen-antibody interaction. Comprising five of the thirteen anchor residues (ASP102, HIS103, SER104, TYR105, and GLN106), the well-known MOG92-106 peptide in complex with the anti-MOG was analysed by AFM and SMD. These analyses evidenced similar force values of 780 pN and 765 pN for computational and experimental MOG92-106 and anti-MOG detachment, respectively. MOG92-106 was responsible for 75% of the total force measured between MOG external domain and anti-MOG, holding the interaction with the antibody. The antigen-antibody binding was confirmed by Surface Plasmon Resonance (SPR) measurements. Combined approaches presented here can conveniently be adjusted to detail novel molecules in diseases research. This can optimize pre-clinical steps, guiding experiments, reducing costs, and animal model usage.

Efeitos do jato de plasma frio sob pressão atmosférica sobre Candida albicans / Cell effects of atmospheric pressure low temperature plasma jet on Candida albicans

As infecções causadas por Candida albicans são consideradas um desafio importante na área médica. Além dos antifúngicos convencionais apresentarem elevada toxicidade, casos de resistência de C. albicans a estes medicamentos têm sido descritos com frequência. Devido a estas limitações do uso de antifúngicos, a busca por terapias alternativas é alvo crescente de estudos. Dentre as novas terapias, o jato de plasma frio destaca-se a como agente eficaz frente a cepas de C. albicans, sendo considerada uma alternativa promissora. Seu efeito antifúngico é associado à presença de espécies reativas de oxigênio e nitrogênio liberadas, assim como de íons e fótons. Todavia até o presente momento, o exato mecanismo de ação não foi elucidado. Portanto, o objetivo deste estudo é prospectar os efeitos do jato de plasma frio sobre C. albicans. Perseguindo este objetivo, foram utilizadas duas técnicas complementares às técnicas microbiológicas clássicas, a espectroscopia no Infra-vermelho (FT-IR) e a microscopia de força atômica (AFM). Suspensões padronizadas da cepa C. albicans SC 5314 e 1 cepa clínica foram expostas ao jato de plasma. A seguir, foram analisadas por espectroscopia no infra-vermelho (FT-IR) e microscopia de força atômica (AFM) para identificar os efeitos do plasma sobre a parede celular de C. albicans. Controle negativo (sem exposição) e positivo (caspofungina) também foram analisados. Os ensaios foram realizados em triplicata em três experimentos independentes e a análise estatística foi realizada ao nível de significância de 5 %. As alterações morfológicas e topográficas nas células tratadas com plasma foram similares às do grupo tratado com caspofungina. O perfil bioquímico das células após tratamento com o plasma foi também similar ao das células tratadas com caspofungina. Conclui-se que o plasma agiu diretamente sobre as estruturas da parede celular fúngica, mais especificamente sobre os polissacarídeos, com provável influencia na redução de glucanos e aumento de mananas e quitinas. Esse remodelamento da parede celular pode ter correlação com as alterações morfológicas observadas, tais como mudança no tamanho da célula e aumento da rugosidade superficial(AU)

Infections caused by Candida albicans are considered important challenges in the medical area. Besides the high toxicity of conventional antifungals, cases of C. albicans resistance have been reported with increasing frequency. Due to these limitations of the antifungals, the search for alternative therapies is increasing. Among these therapies, cold plasma showed effectiveness against C. albicans isolates and is considered a promising alternative. Its antifungal effect is associated to the presence of oxygen and nitrogen reactive species, as well as ions. Nonetheless, the exact mechanism of action on C. albicans has not yet been elucidated. The aim of this study is to investigate the effects of plasma jet on C. albicans cell wall. For this purpose, two techniques complementing to classic the microbiological methods were used, the infrared spectroscopy (FT-IR) and atomic force microscopy (AFM). Standardized suspensions of C. albicans SC 5314 and 1 clinical isolate was exposed to plasma jet. Samples were analyzed by FT-IR and AFM to identifying the effects of plasma on C. albicans. Negative control (no exposition) and positive (caspofungin) were also analyzed. The experiments will be done in triplicate in three independent experiments and the statistical analyzes were done adopting the level of significance of 5%. The morphological and topographic alterations in cells treated with plasma were similar to the group treated with caspofungin. The biochemical profile of the cells after treatment with the plasma jet was also similar to those treated with caspofungin. It could be concluded that plasma acted directly on the structures of fungal cell wall, more specifically on polysaccharides, influencing the reduction of glucans and increased of mannans and chitins. This remodeling of cell wall can be correlated with the detected morphological alterations, such as changes in cell size and increase in superficial roughness(AU)

Infrared Nanospectroscopy Reveals the Chemical Nature of Pit Membranes in Water-Conducting Cells of the Plant Xylem.

In the xylem of angiosperm plants, microscopic pits through the secondary cell walls connect the water-conducting vessels. Cellulosic meshes originated from primary walls, and middle lamella between adjacent vessels, called the pit membrane, separates one conduit from another. The intricate structure of the nano-sized pores in pit membranes enables the passage of water under negative pressure without hydraulic failure due to obstruction by gas bubbles (i.e. embolism) under normal conditions or mild drought stress. Since the chemical composition of pit membranes affects embolism formation and bubble behavior, we directly measured pit membrane composition in Populus nigra wood. Here, we characterized the chemical composition of cell wall structures by synchrotron infrared nanospectroscopy and atomic force microscopy-infrared nanospectroscopy with high spatial resolution. Characteristic peaks of cellulose, phenolic compounds, and proteins were found in the intervessel pit membranes of P. nigra wood. In addition, the vessel to parenchyma pit membranes and developing cell walls of the vascular cambium showed clear signals of cellulose, proteins, and pectin. We did not find a distinct peak of lignin and other compounds in these structures. Our investigation of the complex chemical composition of intervessel pit membranes furthers our understanding of the flow of water and bubbles between neighboring conduits. The advances presented here pave the way for further label-free studies related to the nanochemistry of plant cell components.