Grape pomace and pecan shell fortified bread: The effect of dietary fiber-phenolic compounds interaction on the in vitro accessibility of phenolic compounds and in vitro glycemic index.

Grape pomace (GP) and pecan shell (PS) are two by-products rich in phenolic compounds (PC), and dietary fiber (DF) that may be considered for the development of functional baked foods. In this study, four formulations with different GP:PS ratios (F1(8%:5%), F2(5%:5%), F3(5%:2%), F4(0%:5%), and control bread (CB)) were elaborated and characterized (physiochemical and phytochemical content). Also, their inner structure (SEM), changes in their FTIR functional group's vibrations, and the bioaccessibility of PC and sugars, including an in vitro glycemic index, were analyzed. Results showed that all GP:PS formulations had higher mineral, protein, DF (total, soluble, and insoluble), and PC content than CB. Additionally, PC and non-starch polysaccharides affected gluten and starch absorbance and pores distribution. In vitro digestion model showed a reduction in the glycemic index for all formulations, compared to CB. These findings highlight the possible health benefits of by-products and their interactions in baked goods.

Cryo-Electron Microscopy in the Study of Antiviral Innate Immunity.

Cryo-electron microscopy is a powerful methodology in structural biology and has been broadly used in high-resolution structure determination for challenging samples, which are not readily available for traditional techniques. In particular, the strength of super macro-complexes and the lack of a need for crystals for cryo-EM make this technique feasible for the structural study of complexes involved in antiviral innate immunity. This chapter presents detailed information and experimental procedures of Cryo-EM for determining the structures of the complexes using STING as an example. The procedures included a sample quality check, high-resolution data acquisition, and image processing for Cryo-EM 3D structure determination.

Viability Detection of Foodborne Bacterial Pathogens in Food Environment by PMA-qPCR and by Microscopy Observation.

Foodborne pathogens are responsible for foodborne diseases and food poisoning and thus pose a great threat to food safety. These microorganisms can adhere to surface and form a biofilm composed of an extracellular matrix. This matrix protects bacterial cells from industrial environmental stress factors such as cleaning and disinfection operations. Moreover, during these environmental stresses, many bacterial species can be entered in a viable but nonculturable (VBNC) state. VBNC cells are characterized by an active metabolism and a loss of cultivability on conventional bacteriological agar. This leads to an underestimation of total viable cells in environmental samples and thus may pose a risk for public health. In this chapter, we present a method to detect viable population of foodborne pathogens in industrial environmental samples using a molecular method combining propidium monoazide (PMA) and quantitative PCR (qPCR) and a fluorescence microscopic method associated with the LIVE/DEAD BacLight™ viability stain.

Characterization of Foodborne Pathogens in Biofilms: A Four-Dimensional Structural Dynamics Approach Using HCS-CLSM.

The functional properties of biofilms are intimately related to their spatial architecture. Structural data are therefore of prime importance to dissect the complex social and survival strategies of biofilms and ultimately to improve their control. Confocal laser scanning microscopy (CLSM) is the most widespread microscopic tool to decipher biofilm structure, enabling noninvasive three-dimensional investigation of their dynamics down to the single-cell scale. The emergence of fully automated high content screening (HCS) systems, associated with large-scale image analysis, has radically amplified the flow of available biofilm structural data. In this contribution, we present a HCS-CLSM protocol used to analyze biofilm four-dimensional structural dynamics at high throughput. Meta-analysis of the quantitative variables extracted from HCS-CLSM will contribute to a better biological understanding of biofilm traits.

Zebrafish Larvae Microinjection and Automated Fluorescence Microscopy for Studying Klebsiella pneumoniae Infection and the Host Immune Response.

Studying host-pathogen interactions is essential for understanding infectious diseases and developing possible treatments, especially for priority pathogens with increased virulence and antibiotic resistance, such as Klebsiella pneumoniae. Over time, this subject has been approached from different perspectives, often using mammal host models and invasive endpoint measurements (e.g., sacrifice and organ extraction). However, taking advantage of technological advances, it is now possible to follow the infective process by noninvasive visualization in real time, using optically amenable surrogate hosts. In this line, this chapter describes a live-cell imaging approach to monitor the interaction of K. pneumoniae and potentially other bacterial pathogens with zebrafish larvae in vivo. This methodology is based on the microinjection of fluorescent bacteria into the otic vesicle, followed by time-lapse observation by automated fluorescence microscopy with environmental control, monitoring the dynamics of immune cell recruitment, bacterial load, and larvae survival.

Physicochemical characteristics of airborne microplastics of a typical coastal city in the Yangtze River Delta Region, China.

Airborne microplastics (MPs) are important pollutants that have been present in the environment for many years and are characterized by their universality, persistence, and potential toxicity. This study investigated the effects of terrestrial and marine transport of MPs in the atmosphere of a coastal city and compared the difference between daytime and nighttime. Laser direct infrared imaging (LDIR) and polarized light microscopy were used to characterize the physical and chemical properties of MPs, including number concentration, chemical types, shape, and size. Backward trajectories were used to distinguish the air masses from marine and terrestrial transport. Twenty chemical types were detected by LDIR, with rubber (16.7%) and phenol-formaldehyde resin (PFR; 14.8%) being major components. Three main morphological types of MPs were identified, and fragments (78.1%) are the dominant type. MPs in the atmosphere were concentrated in the small particle size segment (20-50 µm). The concentration of MPs in the air mass from marine transport was 14.7 items/m3 - lower than that from terrestrial transport (32.0 items/m3). The number concentration of airborne MPs was negatively correlated with relative humidity. MPs from terrestrial transport were mainly rubber (20.2%), while those from marine transport were mainly PFR (18%). MPs in the marine transport air mass were more aged and had a lower number concentration than those in the terrestrial transport air mass. The number concentration of airborne MPs is higher during the day than at night. These findings could contribute to the development of targeted control measures and methods to reduce MP pollution.

Perspectives on label-free microscopy of heterogeneous and dynamic biological systems.

Significance: Advancements in label-free microscopy could provide real-time, non-invasive imaging with unique sources of contrast and automated standardized analysis to characterize heterogeneous and dynamic biological processes. These tools would overcome challenges with widely used methods that are destructive (e.g., histology, flow cytometry) or lack cellular resolution (e.g., plate-based assays, whole animal bioluminescence imaging). Aim: This perspective aims to (1) justify the need for label-free microscopy to track heterogeneous cellular functions over time and space within unperturbed systems and (2) recommend improvements regarding instrumentation, image analysis, and image interpretation to address these needs. Approach: Three key research areas (cancer research, autoimmune disease, and tissue and cell engineering) are considered to support the need for label-free microscopy to characterize heterogeneity and dynamics within biological systems. Based on the strengths (e.g., multiple sources of molecular contrast, non-invasive monitoring) and weaknesses (e.g., imaging depth, image interpretation) of several label-free microscopy modalities, improvements for future imaging systems are recommended. Conclusion: Improvements in instrumentation including strategies that increase resolution and imaging speed, standardization and centralization of image analysis tools, and robust data validation and interpretation will expand the applications of label-free microscopy to study heterogeneous and dynamic biological systems.

Quantitative microbiology with widefield microscopy: navigating optical artefacts for accurate interpretations.

Time-resolved live-cell imaging using widefield microscopy is instrumental in quantitative microbiology research. It allows researchers to track and measure the size, shape, and content of individual microbial cells over time. However, the small size of microbial cells poses a significant challenge in interpreting image data, as their dimensions approache that of the microscope's depth of field, and they begin to experience significant diffraction effects. As a result, 2D widefield images of microbial cells contain projected 3D information, blurred by the 3D point spread function. In this study, we employed simulations and targeted experiments to investigate the impact of diffraction and projection on our ability to quantify the size and content of microbial cells from 2D microscopic images. This study points to some new and often unconsidered artefacts resulting from the interplay of projection and diffraction effects, within the context of quantitative microbiology. These artefacts introduce substantial errors and biases in size, fluorescence quantification, and even single-molecule counting, making the elimination of these errors a complex task. Awareness of these artefacts is crucial for designing strategies to accurately interpret micrographs of microbes. To address this, we present new experimental designs and machine learning-based analysis methods that account for these effects, resulting in accurate quantification of microbiological processes.

Tomographic, Biomechanical, and In Vivo Confocal Microscopic Changes in Cornea in Chronic Gout Disease.

PURPOSE: This study aimed to evaluate the tomographic, biomechanical, and in vivo confocal microscopic (IVCM) effects of chronic gout disease on human cornea. METHODS: This prospective study included 60 eyes of 30 participants with chronic gout disease and 60 eyes of 30 healthy controls. Corneal thickness, keratometry (K) readings, and corneal aberrations were measured with Sirius 3 D corneal tomography system (Sirius, CSO, Italy). Corneal biomechanical properties (corneal hysteresis [CH], corneal resistance factor [CRF], and intraocular pressure [IOP] parameters) were assessed with an ocular response analyzer (ORA, Reichert Ophthalmic Instruments). The number and morphology of corneal endothelial cells and the number of basal epithelial cells were evaluated with IVCM (Confoscan 4.0). Tear breakup time (TBUT) was also evaluated. RESULTS: The mean diagnosis time of the patients with gout was 91.2 ± 69.6 months (12-300 month). Among corneal tomography measurements, K readings were similar between the two groups, while total and higher-order aberrations(coma, trefoil,s pherical, and quadrafoil) were significantly higher in the gout group. In the evaluation of biomechanical measurements, the CH value was significantly lower and the corneal-compensated IOP value was significantly higher in the gout group (p = 0.02, p = 0.01, respectively). The two groups did not significantly differ regarding the CRF or Goldmann IOP (p = 0.61, p = 0.15, respectively). Among the IVCM parameters, the number of corneal basal epithelial cells and the percentage of corneal endothelial hexagonality were significantly lower in the gout group, but no significant difference was detected in terms of the number of endothelial cells or polymegathism (p = 0.02, p < 0.001, p = 0.18, p = 0.59, respectively). While TBUT was significantly lower in the gout group (p < 0.001). CONCLUSION: This study showed that chronic gout disease increases the corneal aberrations and decreases the basal epithelial cell count, hexagonality ratio of endothelial cell and corneal biomechanics.

Intercellular Communication Through Microtubular Highways.

Tunneling nanotubes (TNTs) are open-ended, membrane-encased extensions that connect neighboring cells. They have diameters up to 1 µm but are able to expand to convey large cargos. Lengths vary depending on the distance of the cells but have been reported to be capable of extending beyond 300 µm. They have actin cytoskeletons that are essential for their formation, and may or may not have microtubule networks. It is thought that thin TNTs lack microtubules, while thicker TNTs have microtubular highways that use motor proteins to convey materials, including proteins, mitochondria, and nanoparticles between cells. Specifically, the presence of dynein and myosin support trafficking of cargo in both directions. The purpose of these connections is to enable cells to work as a unit or to extend cell life by diluting cytotoxic agents or acquiring biological material needed to survive.

Non-invasive techniques in the clinical diagnosis of cutaneous lymphomas.

In clinical practice, cutaneous lymphomas can be challenging to diagnose or even suspect because they mimic a variety of other inflammatory and neoplastic dermatological conditions. Support for non-invasive skin analysis methods like reflectance confocal microscopy and dermoscopy is still anecdotic. Practically speaking, a deeper and more comprehensive study with a larger number of cases focusing on the effective usefulness of non-invasive techniques should be taken into consideration because they have demonstrated the ability to identify macro and micro features supporting the clinical suspicion of lymphomas, as well as being useful for differential diagnosis and supporting the selection of the biopsy site. The author provides a brief and narrative synopsis of the reflectance confocal microscopy and dermoscopy characteristics of cutaneous lymphomas in this manuscript.

Application of nano-urea in conventional flood-irrigated Boro rice in Bangladesh and nitrogen losses investigation.

Bangladesh stands third in global rice production while complete modernization of rice production is not fully enforced. The boon of nano agriculture might circumvent the challenge of increasing the yield with minimal ecological damage. Nanofertilizer might be one of the solutions to address the problem of modern agriculture confronting environmental hazards owing to the excessive use of synthetic fertilizers by farmers in Bangladesh. We synthesized nanourea by chemical co-precipitation (CP) and hydrothermal (HT) methods in an attempt to develop environmentally friendly nanofertilizers. We characterized the nanourea and confirmed the functionalization of nanohydroxyapatite (nHAP) with urea by scanning transmission electron microscopy (STEM)/EDS mapping. The CP method produced particle dimensions of 45.62 nm for length and 14.16 nm for width. In comparison, the readings obtained through the HT method were around 74.69 nm and 20.44 nm for length and width, respectively. The field application of nanourea demonstrated impressive results, indicating a significant relationship between the particle size of nanourea and its impact on several agricultural factors. The grain yield using traditional synthetic fertilizer (urea) ranged from 6.47 to 6.52 t ha-1 with a very low NUE of 35.8-36.34 %. Contrarily, the grain yield was found from 6.52 to 6.84 t ha-1 and the obtained NUE ranged from 57.58 to 71.0 % using nanourea of the same concentration calibrated with traditional urea by two methods. Additionally, nanourea treatments having 25 % less nitrogen (N) provided higher total N (TN) in grain suggesting possible nutritional enrichment while checking the yield penalty and substantial increase in N use efficiency (NUE). However, further upscaling of this research on a field scale is necessary to confirm the findings.

A novel super-resolution microscopy platform for cutaneous alpha-synuclein detection in Parkinson's disease.

Alpha-synuclein (aSyn) aggregates in the central nervous system are the main pathological hallmark of Parkinson's disease (PD). ASyn aggregates have also been detected in many peripheral tissues, including the skin, thus providing a novel and accessible target tissue for the detection of PD pathology. Still, a well-established validated quantitative biomarker for early diagnosis of PD that also allows for tracking of disease progression remains lacking. The main goal of this research was to characterize aSyn aggregates in skin biopsies as a comparative and quantitative measure for PD pathology. Using direct stochastic optical reconstruction microscopy (dSTORM) and computational tools, we imaged total and phosphorylated-aSyn at the single molecule level in sweat glands and nerve bundles of skin biopsies from healthy controls (HCs) and PD patients. We developed a user-friendly analysis platform that offers a comprehensive toolkit for researchers that combines analysis algorithms and applies a series of cluster analysis algorithms (i.e., DBSCAN and FOCAL) onto dSTORM images. Using this platform, we found a significant decrease in the ratio of the numbers of neuronal marker molecules to phosphorylated-aSyn molecules, suggesting the existence of damaged nerve cells in fibers highly enriched with phosphorylated-aSyn molecules. Furthermore, our analysis found a higher number of aSyn aggregates in PD subjects than in HC subjects, with differences in aggregate size, density, and number of molecules per aggregate. On average, aSyn aggregate radii ranged between 40 and 200 nm and presented an average density of 0.001-0.1 molecules/nm2. Our dSTORM analysis thus highlights the potential of our platform for identifying quantitative characteristics of aSyn distribution in skin biopsies not previously described for PD patients while offering valuable insight into PD pathology by elucidating patient aSyn aggregation status.

Optical microscopy evaluation of root canal filling removal by beginner operators in posterior teeth.

This study analyzed the effectiveness of root canal filling removal in lower molars performed by beginner operators using optical microscopy. A total of 55 mandibular first and second molars with mesial roots exhibiting an average curvature of 10-20° were selected based on preoperative radiographs. Instrumentation was done with ProTaper Gold (Dentsply Sirona) up to F2 (25/.08), using 2ml of 2.5% NaOCl irrigation solution after each file. Root canal obturation was performed using gutta-percha points with cold lateral condensation and Sealapex (Kerr Dental). Coronal fillings were made with composite resin and stored in distilled water for two years. Removal of the root canal fillings was performed with AF Retreatment Rotary (AFRR) and AF Blue R3 (AFBR3) (Fanta Dental Materials) under reciprocating motion with 2.5% NaOCl irrigation. Cross-sections of the coronal, middle, and apical thirds were analyzed at 40x magnification using a STEINDORFF POL microscope with a digital camera. Image analysis was conducted using Image J software, version 1.54, to determine the efficiency of root canal filling removal by percentage. Statistical analysis via one-way ANOVA revealed significant differences between distal and mesial roots (P < 0.05). Specifically, for mesial roots, the removal efficiency was 70.65% in the coronal third, 54.66% in the middle third, and 21.32% in the apical third. Significant difficulties were noted due to fractured files, calcifications, and debris accumulation in the isthmuses. The study concluded that the protocol using Fanta files demonstrated significant differences in removal efficiency correlated with root curvature, compounded by the inexperience of beginner operators. The findings highlight the challenges faced by novice practitioners in achieving effective root canal filling removal.

The 2 stages of cartridge primer toolmark production and the implied impact of cartridge manufacturing tolerances.

Many methods of ballistic toolmark comparison rely upon comparison using 2D greyscale imaging. However, newly emerging analysis methods such as areal surface analysis now utilise an extra dimension of measurement allowing the surface heights/depths of unique toolmark features to be recorded in a densely populated (x,y,z) array for a 3D/areal quantitative comparative analysis. Due to this step change, the colloquialism in referring to the crater produced at the centre of the primer during firing as a "firing pin impression" has become a misnomer, leading some to believe that this toolmark is produced via a single process, where the critical variable is the condition of the firing pin. Furthermore, current forensic ballistic methodology relies on the microscopic differences between individual fired bullets and cartridge cases produced as a result of the manufacturing process of a particular firearm, in this case "matched toolmarks" confirm a ballistic match to a specific firearm. However, very rarely is it considered that the ammunition itself possesses minute differences produced during manufacture that could affect the ballistic match efficacy. This study examines the discharge process of conventional centrefire ammunition and concludes that the unique toolmarks upon the cartridge primer are definitively produced in two defined stages. This conclusion suggests that the factory loading and quality control tolerances of the cartridge itself should now be considered to be a more significant contributing factor to the production of cartridge primer toolmarks than has previously been accepted.

Maximizing the notional area in single tip field emitters.

One of the critical aspects in advancing high-brightness field emitter devices is determining the conditions under which single-tip emitters should be constructed to optimize their emission area. Recent experiments have explored varying the axis ratio ξ of the cap of a single-tip emitter, ranging from an oblate semi-spheroid to a prolate shape, mounted on a nearly cylindrical conducting body. In this work, we present a strategy, based on high-accuracy computer simulations using the finite element technique, to maximize the emission area of those single-tip emitters. Importantly, our findings indicate that the notional emission area achieves its maximum when the emitter's cap is adjusted to an oblate semi-spheroid with a characteristic axis ratio ξC≈0.85. We do a comparison of notional emission area as a function of ξ for an ellipsoidal emitter on a post and compare these results from other emitter configurations, which are feasible to fabricate.

Visualizing highly bright and uniform cellular ultrastructure by expansion-microscopy with tetrahedral DNA nanostructures.

Expansion Microscopy (ExM) is a widely used super-resolution technique that enables imaging of structures beyond the diffraction limit of light. However, ExM suffers from weak labeling signals and expansion distortions, limiting its applicability. Here, we present an innovative approach called Tetrahedral DNA nanostructure Expansion Microscopy (TDN-ExM), addressing these limitations by using tetrahedral DNA nanostructures (TDNs) for fluorescence labeling. Our approach demonstrates a 3- to 10-fold signal amplification due to the multivertex nature of TDNs, allowing the modification of multiple dyes. Previous studies have confirmed minimal distortion on a large scale, and our strategy can reduce the distortion at the ultrastructural level in samples because it does not rely on anchoring agents and is not affected by digestion. This results in a brighter fluorescence, better uniformity, and compatibility with different labeling strategies and optical super-resolution technologies. We validated the utility of TDN-ExM by imaging various biological structures with improved resolutions and signal-to-noise ratios.

Automated High-Throughput Atomic Force Microscopy Single-Cell Nanomechanical Assay Enabled by Deep Learning-Based Optical Image Recognition.

Mechanical forces are essential for life activities, and the mechanical phenotypes of single cells are increasingly gaining attention. Atomic force microscopy (AFM) has been a standard method for single-cell nanomechanical assays, but its efficiency is limited due to its reliance on manual operation. Here, we present a study of deep learning image recognition-assisted AFM that enables automated high-throughput single-cell nanomechanical measurements. On the basis of the label-free identification of the cell structures and the AFM probe in optical bright-field images as well as the consequent automated movement of the sample stage and AFM probe, the AFM probe tip could be accurately and sequentially moved onto the specific parts of individual living cells to perform a single-cell indentation assay or single-cell force spectroscopy in a time-efficient manner. The study illustrates a promising method based on deep learning for achieving operator-independent high-throughput AFM single-cell nanomechanics, which will benefit the application of AFM in mechanobiology.

Protocol for rapid evaluation of antibody-mediated protection in macrophages using immunofluorescence techniques.

Antibodies have been shown to provide protection against tuberculosis (TB). It is important to identify the qualitative and quantitative protective effects of antibodies before a clinical study. Here, we present a protocol to evaluate antibody-mediated protection in macrophages using confocal fluorescence microscopy and flow cytometry. We describe steps for bacteria and macrophage preparation, cell infection, phagocytosis, and phagosome maturation analysis. For complete details on the use and execution of this protocol, please refer to Zeng et al.1.

Micromorphological and Phytochemical Evaluation of Heliotropium rariflorum Stocks From Karak, Pakistan.

Micromorphological and phytochemical studies play a major role in quality control and standardization of traditional or herbal medications. In the present research, micromorphological assessment of Heliotropium rarifloum stocks was performed through light and scanning electron microscopies (LM & SEM). The anatomy of leaves, stem and root showed salient histological features. Both surfaces of the leaves had setose glandular trichomes measuring 20-38 × 6-15 µm. The lower epidermis had comparatively a maximum anomocytic stomata (16-35) and stomatal index (12-33). The mature pollen grains were small (74 µm) and spheroidal shaped, with psilate exine (2 µm) sculpturing. Vein termination and vein islet number of the upper epidermis were 5-20 and 5-15, respectively. The palisade ratio of the leaf lamina for the upper and lower epidermis was 2-10 and 2-8. LM and SEM of the powdered samples displayed crystals, phloem fibers, xylem, vessels, sieve tube elements, companion cells, and tracheids. Extractive values determination, fluorescence, and phytochemical analysis were employed for quality control according to the World Health Organization (WHO) guidelines. Phytochemical screening revealed various secondary metabolites. It is suggested that H. rariflorum might be a reliable source of nutrients and secondary metabolites and might be more medically effective. The current findings confirm its standardization and validation.