Simultaneous Two- and Three-Photon Deep Imaging of Autofluorescence in Bacterial Communities.

The intrinsic fluorescence of bacterial samples has a proven potential for label-free bacterial characterization, monitoring bacterial metabolic functions, and as a mechanism for tracking the transport of relevant components through vesicles. The reduced scattering and axial confinement of the excitation offered by multiphoton imaging can be used to overcome some of the limitations of single-photon excitation (e.g., scattering and out-of-plane photobleaching) to the imaging of bacterial communities. In this work, we demonstrate in vivo multi-photon microscopy imaging of Streptomyces bacterial communities, based on the excitation of blue endogenous fluorophores, using an ultrafast Yb-fiber laser amplifier. Its parameters, such as the pulse energy, duration, wavelength, and repetition rate, enable in vivo multicolor imaging with a single source through the simultaneous two- and three-photon excitation of different fluorophores. Three-photon excitation at 1040 nm allows fluorophores with blue and green emission spectra to be addressed (and their corresponding ultraviolet and blue single-photon excitation wavelengths, respectively), and two-photon excitation at the same wavelength allows fluorophores with yellow, orange, or red emission spectra to be addressed (and their corresponding green, yellow, and orange single-photon excitation wavelengths). We demonstrate that three-photon excitation allows imaging over a depth range of more than 6 effective attenuation lengths to take place, corresponding to an 800 micrometer depth of imaging, in samples with a high density of fluorescent structures.

Non-Destructive Direct Pericarp Thickness Measurement of Sorghum Kernels Using Extended-Focus Optical Coherence Microscopy.

Non-destructive measurements of internal morphological structures in plant materials such as seeds are of high interest in agricultural research. The estimation of pericarp thickness is important to understand the grain quality and storage stability of seeds and can play a crucial role in improving crop yield. In this study, we demonstrate the applicability of fiber-based Bessel beam Fourier domain (FD) optical coherence microscopy (OCM) with a nearly constant high lateral resolution maintained at over ~400 µm for direct non-invasive measurement of the pericarp thickness of two different sorghum genotypes. Whereas measurements based on axial profiles need additional knowledge of the pericarp refractive index, en-face views allow for direct distance measurements. We directly determine pericarp thickness from lateral sections with a 3 µm resolution by taking the width of the signal corresponding to the pericarp at the 1/e threshold. These measurements enable differentiation of the two genotypes with 100% accuracy. We find that trading image resolution for acquisition speed and view size reduces the classification accuracy. Average pericarp thicknesses of 74 µm (thick phenotype) and 43 µm (thin phenotype) are obtained from high-resolution lateral sections, and are in good agreement with previously reported measurements of the same genotypes. Extracting the morphological features of plant seeds using Bessel beam FD-OCM is expected to provide valuable information to the food processing industry and plant breeding programs.

Erratum: Altomare et al. In-Depth AGE and ALE Profiling of Human Albumin in Heart Failure: Ex Vivo Studies. Antioxidants 2021, 10, 358.

The authors of this paper [...].

Extended focal depth Fourier domain optical coherence microscopy with a Bessel-beam - LP02 mode - from a higher order mode fiber.

We present a robust fiber-based setup for Bessel-like beam extended depth-of-focus Fourier-domain optical coherence microscopy, where the Bessel-like beam is generated in a higher order mode fiber module. In this module a stable guided LP02 core mode is selectively excited by a long period grating written in the higher order mode fiber. Imaging performance of this system in terms of lateral resolution and depth of focus was analyzed using samples of suspended microbeads and compared to the case where illumination is provided by the fundamental LP01 mode of a single mode fiber. Illumination with the LP02 mode allowed for a lateral resolution down to 2.5 µm as compared to 4.5 µm achieved with the LP01 mode of the single mode fiber. A three-fold enhancement of the depth of focus compared to a Gaussian beam with equally tight focus is achieved with the LP02 mode. Analysis of the theoretical lateral point spread functions for the case of LP01 and LP02 illumination agrees well with the experimental data. As the design space of waveguides and long-period gratings allows for further optimization of the beam parameters of the generated Bessel-like beams in an all-fiber module, this approach offers a robust and yet flexible alternative to free-space optics approaches or the use of conical fiber tips.

Lipid Peroxidation in Atherosclerotic Cardiovascular Diseases.

Significance: Atherosclerotic cardiovascular diseases (ACVDs) continue to be a primary cause of mortality worldwide in adults aged 35-70 years, occurring more often in countries with lower economic development, and they constitute an ever-growing global burden that has a considerable socioeconomic impact on society. The ACVDs encompass diverse pathologies such as coronary artery disease and heart failure (HF), among others. Recent Advances: It is known that oxidative stress plays a relevant role in ACVDs and some of its effects are mediated by lipid oxidation. In particular, lipid peroxidation (LPO) is a process under which oxidants such as reactive oxygen species attack unsaturated lipids, generating a wide array of oxidation products. These molecules can interact with circulating lipoproteins, to diffuse inside the cell and even to cross biological membranes, modifying target nucleophilic sites within biomolecules such as DNA, lipids, and proteins, and resulting in a plethora of biological effects. Critical Issues: This review summarizes the evidence of the effect of LPO in the development and progression of atherosclerosis-based diseases, HF, and other cardiovascular diseases, highlighting the role of protein adduct formation. Moreover, potential therapeutic strategies targeted at lipoxidation in ACVDs are also discussed. Future Directions: The identification of valid biomarkers for the detection of lipoxidation products and adducts may provide insights into the improvement of the cardiovascular risk stratification of patients and the development of therapeutic strategies against the oxidative effects that can then be applied within a clinical setting.

S-Thiolation Targets Albumin in Heart Failure.

Human serum albumin (HSA) is associated with several physiological functions, such as maintaining oncotic pressure and microvascular integrity, among others. It also represents the major and predominant antioxidant in plasma due to the presence of the Cys34 sulfhydryl group. In this study, we assessed qualitative and quantitative changes in HSA in patients with heart failure (HF) and their relationship with the severity of the disease. We detected by means of mass spectrometry a global decrease of the HSA content in the plasma of HF patients in respect to control subjects, a significant increase of thio-HSA with a concomitant decrease in the reduced form of albumin. Cysteine and, at a lesser extent, homocysteine represent the most abundant thiol bound to HSA. A strong inverse correlation was also observed between cysteine-HSA and peak VO2/kg, an index of oxygen consumption associated with HF severity. Moreover, in HL-1 cardiomyocytes incubated with H2O2, we showed a significant decrease of cell viability in cells treated with thio-HSA in respect to restored native-HSA. In conclusion, we found for the first time that S-thiolation of albumin is increased in the plasma of HF patients and induced changes in the structure and antioxidant function of HSA, likely contributing to HF progression.

Pro-oxidant and pro-inflammatory effects of glycated albumin on cardiomyocytes.

Human serum albumin (HSA) is the most abundant circulating protein in the body and presents an extensive range of biological functions. As such, it is prone to undergo post-translational modifications (PTMs). The non-enzymatic early glycation of HSA, one of the several PTMs undergone by HSA, arises from the addition of reducing sugars to amine group residues, thus modifying the structure of HSA. These changes may affect HSA functions impairing its biological activity, finally leading to cell damage. The aim of this study was to quantitate glycated-HSA (GA) levels in the plasma of heart failure (HF) patients and to evaluate the biological effects of GA on HL-1 cardiomyocytes. Plasma GA content from HF patients and healthy subjects was measured by direct infusion electrospray ionization mass spectrometry (ESI-MS). Results pointed out a significant increase of GA in HF patients with respect to the control group (p < 0.05). Additionally, after stimulation with GA, proteomic analysis of HL-1 secreted proteins showed the modulation of several proteins involved, among other processes, in the response to stress. Further, stimulated cells showed a rapid increase in ROS generation, higher mRNA levels of the inflammatory cytokine interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α), and higher levels of the oxidative 4-HNE-protein adducts and carbonylated proteins. Our findings show that plasma GA is increased in HF patients. Further, GA exerts pro-inflammatory and pro-oxidant effects on cardiomyocytes, which suggest a causal role in the etiopathogenesis of HF.

Lipoxidation in cardiovascular diseases.

Lipids can go through lipid peroxidation, an endogenous chain reaction that consists in the oxidative degradation of lipids leading to the generation of a wide variety of highly reactive carbonyl species (RCS), such as short-chain carbonyl derivatives and oxidized truncated phospholipids. RCS exert a wide range of biological effects due to their ability to interact and covalently bind to nucleophilic groups on other macromolecules, such as nucleic acids, phospholipids, and proteins, forming reversible and/or irreversible modifications and generating the so-called advanced lipoxidation end-products (ALEs). Lipoxidation plays a relevant role in the onset of cardiovascular diseases (CVD), mainly in the atherosclerosis-based diseases in which oxidized lipids and their adducts have been extensively characterized and associated with several processes responsible for the onset and development of atherosclerosis, such as endothelial dysfunction and inflammation. Herein we will review the current knowledge on the sources of lipids that undergo oxidation in the context of cardiovascular diseases, both from the bloodstream and tissues, and the methods for detection, characterization, and quantitation of their oxidative products and protein adducts. Moreover, lipoxidation and ALEs have been associated with many oxidative-based diseases, including CVD, not only as potential biomarkers but also as therapeutic targets. Indeed, several therapeutic strategies, acting at different levels of the ALEs cascade, have been proposed, essentially blocking ALEs formation, but also their catabolism or the resulting biological responses they induce. However, a deeper understanding of the mechanisms of formation and targets of ALEs could expand the available therapeutic strategies.

An Emergent Entity: Indolent Mucormycosis of the Paranasal Sinuses. A Multicenter Study

Abstract Introduction Indolent or chronic mucormycosis is a rare entity that affects both immunosuppressed and immunocompetent individuals. Additionally, its clinical evolution is nonspecific and there is no standardized treatment for this condition. Objective To describe the clinical characteristics and management of patients with indolent mucormycosis. Methods In the project of study with chart review in the Interinstitutional secondary care centers, patients with evidence of indolentmucormycosis, defined as pathological confirmation of nasal/paranasal sinus mucormycosis for more than 1 month, were included. All patients underwent complete laboratory workup, imaging studies, surgical treatment and adequate follow-up. No evidence of disease status was defined when patient had subsequent biopsies with no evidence of mucormycosis. Results We included seven patients, three female and four male subjects. The mean age was 53.14 years. Four patients were immunosuppressed and three immunocompetent. Among the immunosuppressed patients three had diabetes and one had dermatomyositis. The symptomswere nonspecific: facial pain/headache, mucoid discharge and cacosmiawere the ones most frequently reported. Maxillary sinus involvement was present in all patients. Two immunosuppressed subjects received amphotericin. Posaconazole was the only treatmentinoneimmunosuppressedpatient. Allimmunocompetent patientshadsingleparanasal sinus disease and received only surgical treatment. All patients are alive and free of disease. Conclusion Indolent mucormycosis is a new and emerging clinical entity in immunosuppressed and immunocompetent patients. Single paranasal sinus disease is a frequent presentation and should not be overlooked as a differential diagnosis in these patients. Immunocompetent patients should only be treated surgically. (AU)

An Emergent Entity: Indolent Mucormycosis of the Paranasal Sinuses. A Multicenter Study.

Introduction Indolent or chronic mucormycosis is a rare entity that affects both immunosuppressed and immunocompetent individuals. Additionally, its clinical evolution is nonspecific and there is no standardized treatment for this condition. Objective To describe the clinical characteristics and management of patients with indolent mucormycosis. Methods In the project of study with chart review in the Interinstitutional secondary care centers, patients with evidence of indolent mucormycosis, defined as pathological confirmation of nasal/paranasal sinus mucormycosis for more than 1 month, were included. All patients underwent complete laboratory workup, imaging studies, surgical treatment and adequate follow-up. No evidence of disease status was defined when patient had subsequent biopsies with no evidence of mucormycosis. Results We included seven patients, three female and four male subjects. The mean age was 53.14 years. Four patients were immunosuppressed and three immunocompetent. Among the immunosuppressed patients three had diabetes and one had dermatomyositis. The symptoms were nonspecific: facial pain/headache, mucoid discharge and cacosmia were the ones most frequently reported. Maxillary sinus involvement was present in all patients. Two immunosuppressed subjects received amphotericin. Posaconazole was the only treatment in one immunosuppressed patient. All immunocompetent patients had single paranasal sinus disease and received only surgical treatment. All patients are alive and free of disease. Conclusion Indolent mucormycosis is a new and emerging clinical entity in immunosuppressed and immunocompetent patients. Single paranasal sinus disease is a frequent presentation and should not be overlooked as a differential diagnosis in these patients. Immunocompetent patients should only be treated surgically.

Reprint of: Proteomics in cardiovascular diseases: Unveiling sex and gender differences in the era of precision medicine.

Cardiovascular diseases (CVDs) represent the most important cause of mortality in women and in men. Contrary to the long-standing notion that the effects of the major risk factors on CVD outcomes are the same in both sexes, recent evidence recognizes new, potentially independent, sex/gender-related risk factors for CVDs, and sex/gender-differences in the clinical presentation of CVDs have been demonstrated. Furthermore, some therapeutic options may not be equally effective and safe in men and women. In this context, proteomics offers an extremely useful and versatile analytical platform for biomedical researches that expand from the screening of early diagnostic and prognostic biomarkers to the investigation of the molecular mechanisms underlying CDVs. In this review, we summarized the current applications of proteomics in the cardiovascular field, with emphasis on sex and gender-related differences in CVDs. SIGNIFICANCE: Increasing evidence supports the profound effect of sex and gender on cardiovascular physio-pathology and the response to drugs. A clear understanding of the mechanisms underlying sexual dimorphisms in CVDs would not only improve our knowledge of the etiology of these diseases, but could also inform health policy makers and guideline committees in tailoring specific interventions for the prevention, treatment and management of CVDs in both men and women.

Proteomics in cardiovascular diseases: Unveiling sex and gender differences in the era of precision medicine.

Cardiovascular diseases (CVDs) represent the most important cause of mortality in women and in men. Contrary to the long-standing notion that the effects of the major risk factors on CVD outcomes are the same in both sexes, recent evidence recognizes new, potentially independent, sex/gender-related risk factors for CVDs, and sex/gender-differences in the clinical presentation of CVDs have been demonstrated. Furthermore, some therapeutic options may not be equally effective and safe in men and women. In this context, proteomics offers an extremely useful and versatile analytical platform for biomedical researches that expand from the screening of early diagnostic and prognostic biomarkers to the investigation of the molecular mechanisms underlying CDVs. In this review, we summarized the current applications of proteomics in the cardiovascular field, with emphasis on sex and gender-related differences in CVDs. SIGNIFICANCE: Increasing evidence supports the profound effect of sex and gender on cardiovascular physio-pathology and the response to drugs. A clear understanding of the mechanisms underlying sexual dimorphisms in CVDs would not only improve our knowledge of the etiology of these diseases, but could also inform health policy makers and guideline committees in tailoring specific interventions for the prevention, treatment and management of CVDs in both men and women.

Exploring the biochemistry of the prenylome and its role in disease through proteomics: progress and potential.

INTRODUCTION: Protein prenylation is a ubiquitous covalent post-translational modification characterized by the addition of farnesyl or geranylgeranyl isoprenoid groups to a cysteine residue located near the carboxyl terminal of a protein. It is essential for the proper localization and cellular activity of numerous proteins, including Ras family GTPases and G-proteins. In addition to its roles in cellular physiology, the prenylation process has important implications in human diseases and in the recent years, it has become attractive target of inhibitors with therapeutic potential. Areas covered: This review attempts to summarize the basic aspects of prenylation integrating them with biological functions in diseases and giving an account of the current status of prenylation inhibitors as potential therapeutics. We also summarize the methodologies for the characterization of this modification. Expert commentary: The growing body of evidence suggesting an important role of prenylation in diseases and the subsequent development of inhibitors of the enzymes responsible for this modification lead to the urgent need to identify the full spectrum of prenylated proteins that are altered in the disease or affected by drugs. Proteomic tools to analyze prenylated proteins are recently emerging, thanks to the advancement in the field of mass spectrometry coupled to enrichment strategies.

Fast volumetric calcium imaging across multiple cortical layers using sculpted light.

Although whole-organism calcium imaging in small and semi-transparent animals has been demonstrated, capturing the functional dynamics of large-scale neuronal circuits in awake behaving mammals at high speed and resolution has remained one of the main frontiers in systems neuroscience. Here we present a method based on light sculpting that enables unbiased single- and dual-plane high-speed (up to 160 Hz) calcium imaging as well as in vivo volumetric calcium imaging of a mouse cortical column (0.5 mm × 0.5 mm × 0.5 mm) at single-cell resolution and fast volume rates (3-6 Hz). We achieved this by tailoring the point-spread function of our microscope to the structures of interest while maximizing the signal-to-noise ratio using a home-built fiber laser amplifier with pulses that are synchronized to the imaging voxel speed. This enabled in vivo recording of calcium dynamics of several thousand neurons across cortical layers and in the hippocampus of awake behaving mice.

High-order dispersion in chirped-pulse oscillators.

The effects of high-order dispersion on a chirped-pulse oscillator operating in the positive dispersion regime were studied both theoretically and experimentally. It was found that odd and negative even high-order dispersions impair the oscillator stability owing to resonance with the dispersion waves, but can broaden the spectrum as in the case of continuum generation in the fibers. Positive fourth-order dispersion enhances the stability and shifts the stability range into negative dispersion. The destabilization mechanism was found to be a parametrical instability which causes noisy mode locking around zero dispersion.

Daily intake of 100 mg ascorbic acid as urinary tract infection prophylactic agent during pregnancy.

OBJECTIVE: To evaluate the role a daily intake of 100 mg of ascorbic acid plays in urinary infection prophylaxis during pregnancy. METHODS AND MATERIALS: A single-blind clinical trial was carried out on pregnant women randomly assigned to the following treatment groups - Group A: oral treatment with ferrous sulphate (200 mg per day), folic acid (5 mg per day) and ascorbic acid (100 mg per day) for 3 months, and Group B: oral treatment with ferrous sulphate (200 mg per day) and folic acid (5 mg per day) for 3 months. All patients were clinically evaluated, and a urine culture was carried out each month for a period of 3 months. The chi(2) and odds ratio were used to compare effects with and without ascorbic acid, and statistical significance was considered at p<0.05. RESULTS: Global frequency of urinary infections was 25%. The presence of urinary infections in Group A (12.7%) was significantly lower than in Group B (29.1%), (p=0.03, OR =0.35, CI 95% =0.13-0.91). CONCLUSIONS: Daily intake of 100 mg of ascorbic acid played an important role in the reduction of urinary infections, improving the health level of the gestating women. We recommend additional vitamin C intake for pregnant women in populations which have a high incidence of bacteriuria and urinary infections.

Few-cycle carrier envelope phase-dependent stereo detection of electrons.

The spatial distribution of electrons emitted from atoms by few-cycle optical fields is known to be dependent on the carrier envelope phase, i.e., the phase of the field with respect to the pulse envelope. With respect to Paulus et al. [Phys. Rev. Lett.91, 253004 (2003)] we propose a greatly simplified device to measure and control the carrier envelope phase of few-cycle pulses with an accuracy of better than pi/10 based on this principle. We compared different schemes to control the carrier envelope phase of our pulses.