Analysis of Neurite and Spine Formation in Neurons In Vitro.

Primary neuronal cultures are commonly used to study genetic and exogenous factors influencing neuronal development and maturation. During development, neurons undergo robust morphological changes involving expansion of dendritic arbor, formation of dendritic spines, and expression of synaptic proteins. In this chapter, we will cover methodological approaches allowing quantitative assessment of in vitro cultured neurons. Various quantitative characteristics of dendritic arbor can be derived based on immunostaining against anti-microtubule-associated protein 2 followed by dendrite tracing with the SNT plug-in of the FIJI software package. The number and subtypes of dendritic spines can be assessed by double labeling with DiI and Phalloidin iFluor448 followed by laser scanning confocal microscopy analysis. Finally, expression of presynaptic and postsynaptic proteins can be determined by immunohistochemistry and quantification using several available software packages including FIJI and Imaris, which also allows for 3D rendering and statistical displaying of the expression level of synaptic proteins.

Novel Techniques for Mapping DNA Damage and Repair in the Brain.

DNA damage in the brain is influenced by endogenous processes and metabolism along with exogenous exposures. Accumulation of DNA damage in the brain can contribute to various neurological disorders, including neurodegenerative diseases and neuropsychiatric disorders. Traditional methods for assessing DNA damage in the brain, such as immunohistochemistry and mass spectrometry, have provided valuable insights but are limited by their inability to map specific DNA adducts and regional distributions within the brain or genome. Recent advancements in DNA damage detection methods offer new opportunities to address these limitations and further our understanding of DNA damage and repair in the brain. Here, we review emerging techniques offering more precise and sensitive ways to detect and quantify DNA lesions in the brain or neural cells. We highlight the advancements and applications of these techniques and discuss their potential for determining the role of DNA damage in neurological disease.

Assessing Phagocytosis of Cryptococcus neoformans Cells in Human Monocytes or the J774 Murine Macrophage Cell Line.

Monocyte/macrophage cells play a central role in innate immunity against C. neoformans and C. gattii, species known to cause human disease. Cryptococcus is the only fungal genus known to possess such a large extracellular polysaccharide capsule, which impacts interactions of innate cells with the yeast. This interaction results in different fates, such as phagocytosis and intracellular proliferation and, as the interaction progresses, vomocytosis, cell-to-cell transfer, lysis of macrophages, or yeast killing. Differentiating internalized versus external Cryptococcus cells is thus essential to evaluate monocyte-macrophage phagocytosis. We describe here a protocol that allows quantification of Cryptococcus spp. phagocytosis using quantitative flow cytometry in human monocytes and a murine macrophage cell line (J774).

Emergent antibiotic persistence in a spatially structured synthetic microbial mutualism.

Antibiotic persistence (heterotolerance) allows a subpopulation of bacteria to survive antibiotic-induced killing and contributes to the evolution of antibiotic resistance. Although bacteria typically live in microbial communities with complex ecological interactions, little is known about how microbial ecology affects antibiotic persistence. Here, we demonstrated within a synthetic two-species microbial mutualism of Escherichia coli and Salmonella enterica that the combination of cross-feeding and community spatial structure can emergently cause high antibiotic persistence in bacteria by increasing the cell-to-cell heterogeneity. Tracking ampicillin-induced death for bacteria on agar surfaces, we found that E. coli forms up to 55 times more antibiotic persisters in the cross-feeding coculture than in monoculture. This high persistence could not be explained solely by the presence of S. enterica, the presence of cross-feeding, average nutrient starvation, or spontaneous resistant mutations. Time-series fluorescent microscopy revealed increased cell-to-cell variation in E. coli lag time in the mutualistic co-culture. Furthermore, we discovered that an E. coli cell can survive antibiotic killing if the nearby S. enterica cells on which it relies die first. In conclusion, we showed that the high antibiotic persistence phenotype can be an emergent phenomenon caused by a combination of cross-feeding and spatial structure. Our work highlights the importance of considering spatially structured interactions during antibiotic treatment and understanding microbial community resilience more broadly.

Biological Efficacy of Ionizing Radiation Sources on 3D Organotypic Tissue Slices Assessed by Fluorescence Microscopy.

OBJECTIVE: Traditional cell-based radiobiological methods are inadequate for assessing the toxicity of ionizing radiation exposure in relation to the microstructure of the extracellular matrix. Organotypic tissue slices preserve the spatial organization observed in vivo, making the tissue easily accessible for visualization and staining. This study aims to explore the use of fluorescence microscopy of physiologically compatible 3D tissue cultures to assess the effects of ionizing radiation. METHODS: Organotypic tissue slices were obtained by vibratome, and their mechanical properties were studied. Slices were exposed by two ionizing radiation sources; electron beams (80 Gy and 4 Gy), and soft gamma irradiation (80 Gy and 4 Gy). Two tissue culture protocols were used: the standard (37°C), and hypothermic (30°C) conditions. A qualitative analysis of cell viability in organotypic tissue slices was performed using fluorescent dyes and standard laser confocal microscopy. RESULTS: Biological dosimetry is represented by differentially stained 200-µm thick organotypic tissue sections related to living and dead cells and cell metabolic activity. CONCLUSION: Our results underscore the ability of fluorescence laser scanning confocal microscopy to rapidly assess the radiobiological effects of ionizing radiation in vitro on 3D organotypic tissue slices.

Selective visual staining of polyurethane microplastics by novel colorimetric and near-infrared (NIR) fluorescent dye: Application to environmental water and natural soil samples.

Microplastics can cause environmental pollution and ecosystem destruction as well as human health problems. Among the types of microplastics, polyurethane (PU) is particularly resistant to heat and difficult to decompose, causing disposal problems, and is evaluated as one of the most hazardous polymers. We present a novel colorimetric and near-infrared (NIR) fluorescence dye, (E)-N-(2-((4-(diphenylamino)benzylidene)amino)phenyl)- 7-nitrobenzo[c][1,2,5]oxadiazol-4-amine (DPNA), designed for selective visual PU microplastic staining. The intramolecular charge transfer (ICT) properties of DPNA are demonstrated through density functional theory (DFT) calculations along with solvatochromic shift. DPNA exhibits red color and red fluorescence emission, showing promising potential as a staining dye. To achieve selective PU microplastic staining, we establish an optimized experimental procedure with the staining dye DPNA by evaluating the staining efficiency under different staining solvent compositions and staining times. DPNA can distinguish PU by both red fluorescence signal and red coloration among different types of microplastics. In addition, DPNA well stain fresh PUs with diverse sizes and at various pH range of 5-9, and the aged PUs can also be dyed as effectively as the fresh PU. Most importantly, DPNA selectively stains PU among 11 types of microplastics and 5 types of natural particles in environmental water and soil with and without any pre-treatments. The adsorption mechanism of DPNA on PU microplastic is demonstrated through field emission scanning electron microscopes (FE-SEM), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), X-ray photoelectron spectroscopy (XPS), and non-covalent interaction (NCI)-reduced density gradient (RDG) analyses, and proposed that intermolecular hydrogen bonding has a significant effect.

Insight into the molecular mechanism, cytotoxic, and anticancer activities of phyto-reduced silver nanoparticles in MCF-7 breast cancer cell lines.

This contribution insight on the cytotoxic and anticancer activities and molecular mechanism of phyto-reduced silver nanoparticles (AgNPs) in MCF-7 breast cancer cell lines. A simple, entirely green synthesis process was optimized for the phyto-reduction of AgNP (~12.7 nm) using aqueous leaf extracts of Indigofera heterantha. The structural and vibrational properties of biosynthesized AgNPs were extensively characterized using UV-Vis spectrophotometer, x-ray diffraction (XRD), dynamic light scattering (DLS), and Fourier transform Infrared spectroscopy (FTIR), while their shape and morphology was studied through scanning electron microscopy (SEM). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay indicates concentration dependent inhibition with IC50, 27.93 ± 2.10 µg/mL against MCF-7 cells and 294.38 ± 3.87 µg/mL against L929 cells. The manifested anticancer mechanism in MCF-7 cells was extensively studied using Acridine orange/ethidium bromide (AO/EB), 4',6-diamidino-2-phenylindole (DAPI) and Annexin-V/propedium iodide fluorescence microscopic assays. The level of reactive oxygen species (ROS) was measured using DCFH-DA fluorescent spectroscopy. Overall, the results show that AgNPs exhibit cytotoxic and apoptotic effect on breast cancer MCF-7 cells by damaging membrane integrity and nuclear fragmentation due to oxidative stress generated by elevated level of ROS. RESEARCH HIGHLIGHTS: Biomimetic synthesis of nano dimension size silver nanoparticles (AgNPs). Characterization of AgNPs through UV-Vis, DLS, XRD, FTIR, and SEM. Cytotoxic and anticancer effects of the biosynthesized AgNPs in L929 fibroblast cells and MCF7 breast cancer respectively. Determination of morphological, and nuclear changes triggered by AgNPs in MCF 7 breast cancer cells using fluorescent microscopy and flow cytometry. Apoptosis induction by AgNPs in cancer cells through oxidative stress generated by reactive oxygen species (ROS).

Glymphatic fluid transport is suppressed by the aquaporin-4 inhibitor AER-271.

The glymphatic system transports cerebrospinal fluid (CSF) into the brain via arterial perivascular spaces and removes interstitial fluid from the brain along perivenous spaces and white matter tracts. This directional fluid flow supports the clearance of metabolic wastes produced by the brain. Glymphatic fluid transport is facilitated by aquaporin-4 (AQP4) water channels, which are enriched in the astrocytic vascular endfeet comprising the outer boundary of the perivascular space. Yet, prior studies of AQP4 function have relied on genetic models, or correlated altered AQP4 expression with glymphatic flow in disease states. Herein, we sought to pharmacologically manipulate AQP4 function with the inhibitor AER-271 to assess the contribution of AQP4 to glymphatic fluid transport in mouse brain. Administration of AER-271 inhibited glymphatic influx as measured by CSF tracer infused into the cisterna magna and inhibited increases in the interstitial fluid volume as measured by diffusion-weighted MRI. Furthermore, AER-271 inhibited glymphatic efflux as assessed by an in vivo clearance assay. Importantly, AER-271 did not affect AQP4 localization to the astrocytic endfeet, nor have any effect in AQP4 deficient mice. Since acute pharmacological inhibition of AQP4 directly decreased glymphatic flow in wild-type but not in AQP4 deficient mice, we foresee AER-271 as a new tool for manipulation of the glymphatic system in rodent brain.

Live Imaging Transverse Sections of Zebrafish Embryo Explants.

Vertebrate embryogenesis is a highly dynamic process involving coordinated cell and tissue movements that generate the final embryonic body plan. Many of these movements are difficult to image at high resolution because they occur deep within the embryo along the midline, causing light scattering and requiring longer working distances. Here, we present an explant-based method to image transverse cross sections of living zebrafish embryos. This method allows for the capture of all cell movements at high-resolution throughout the embryonic trunk, including hard-to-image deep tissues. This technique offers an alternative to expensive or computationally difficult microscopy methods. Key features • Generates intact zebrafish explants with minimal tissue disturbance. • Allows for live imaging of deep tissues normally obscured by common confocal microscopy techniques. • Immobilizes tissues for extended periods required for time-lapse imaging. • Utilizes readily available reagents and tools, which can minimize the time and cost of the procedure.

Microscopy methods for Clostridioides difficile.

Microscopic technologies including light and fluorescent, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and cryo-electron microscopy have been widely utilized to visualize Clostridioides difficile at the molecular, cellular, community, and structural biology level. This comprehensive review summarizes the microscopy tools (fluorescent and reporter system) in their use to study different aspects of C. difficile life cycle and virulence (sporulation, germination) or applications (detection of C. difficile or use of antimicrobials). With these developing techniques, microscopy tools will be able to find broader applications and address more challenging questions to study C. difficile and C. difficile infection.

Interactive effects of cyanobacterial metabolites aeruginosin-98B, anabaenopeptin-B and cylindrospermopsin on physiological parameters and novel in vivo fluorescent indicators in Chironomus aprilinus larvae.

We aimed to determine the effects of single cyanobacterial metabolites aeruginosin-B (AER-B), anabaenopeptin-B (ANA-B), cylindrospermopsin (CYL), their binary and ternary mixtures on biomarkers of Chironomus aprilinus larvae: oxygen consumption, fat body structure and two novel fluorescent indicators: imaging of nuclei in cells of body integument, and the catecholamine level. The obtained results showed that oxygen consumption was inhibited by single tested cyanobacterial metabolites except for ANA-B at the lowest concentration (250 µg/L). Although the mixtures of the metabolites inhibited oxygen consumption with antagonistic interactions between the components stimulation was noted in the group exposed to the lowest concentrations of AER-B + CYL (125 µg/L + 125 µg/L, respectively) and the ternary mixture of AER-B + ANA-B + CYL (83.3 µg/L + 83.3 µg/L + 83.3 µg/L, respectively). In vivo fluorescent staining with Hoechst 34580 showed that single AER-B had lower cytotoxic potential on body integument cells than ANA-B and CYL and most binary mixtures except for AER-B + CYL induced synergistic toxicity. Catecholamine level was decreased in animals exposed to single metabolites, their binary and ternary mixtures; however, the interactions between the components in the ternary mixture were antagonistic. Fat body was found to be disrupted in the larvae exposed to single metabolites and their combinations. Antagonistic toxic interactions between the oligopeptide components were found in most binary and the ternary mixtures; however, synergistic effect was noted in the binary mixture of AER-B + CYL. The results suggest that in natural conditions Chironomus larvae and possibly other benthic invertebrates may be affected by cyanobacterial metabolites, however various components and in mixtures and their concentrations may determine varied physiological effects and diverse interactions.

Assembly of phiKZ bacteriophage Inner Body during infection.

The giant myovirus phiKZ is characterised by an Inner Body (IB) structure within its capsid, crucial for orderly DNA packaging. The IB is composed of six phiKZ-specific proteins. Notably, four of these IB proteins are co-injected with DNA into the host cell, where they potentially play a role in attacking the bacterial cell. The dynamics of IB assembling within the phiKZ capsid during infection remain poorly understood. In this study, we used fluorescent microscopy to track the localisation of IB proteins fused to fluorescent proteins within the cell throughout the infection process. Our findings reveal that the proteins Gp97 and Gp162 are incorporated into new virion heads during phage head maturation. In contrast, proteins Gp90, Gp93, and Gp95 are likely integrated into the virion shortly before the DNA packaging.

Electric organ discharge from electric eel facilitates DNA transformation into teleost larvae in laboratory conditions.

Background: Electric eels (Electrophorus sp.) are known for their ability to produce electric organ discharge (EOD) reaching voltages of up to 860 V. Given that gene transfer via intense electrical pulses is a well-established technique in genetic engineering, we hypothesized that electric eels could potentially function as a gene transfer mechanism in their aquatic environment. Methods: To investigate this hypothesis, we immersed zebrafish larvae in water containing DNA encoding the green fluorescent protein (GFP) and exposed them to electric eel's EOD. Results and Discussion: Some embryos exhibited a mosaic expression of green fluorescence, in contrast to the control group without electrical stimulation, which showed little distinct fluorescence. This suggests that electric eel EOD has the potential to function as an electroporator for the transfer of DNA into eukaryotic cells. While electric eel EOD is primarily associated with behaviors related to sensing, predation, and defense, it may incidentally serve as a possible mechanism for gene transfer in natural environment. This investigation represents the initial exploration of the uncharted impact of electric eel EOD, but it does not directly establish its significance within the natural environment. Further research is required to understand the ecological implications of this phenomenon.

Annexin A5 stabilizes matrix vesicle-biomimetic lipid membranes: unravelling a new role of annexins in calcification.

Matrix vesicles are a special class of extracellular vesicles thought to actively contribute to both physiologic and pathologic mineralization. Proteomic studies have shown that matrix vesicles possess high amounts of annexin A5, suggesting that the protein might have multiple roles at the sites of calcification. Currently, Annexin A5 is thought to promote the nucleation of apatitic minerals close to the inner leaflet of the matrix vesicles' membrane enriched in phosphatidylserine and Ca2+. Herein, we aimed at unravelling a possible additional role of annexin A5 by investigating the ability of annexin A5 to adsorb on matrix-vesicle biomimetic liposomes and Langmuir monolayers made of dipalmitoylphosphatidylserine (DPPS) and dipalmitoylphosphatidylcholine (DPPC) in the absence and in the presence of Ca2+. Differential scanning calorimetry and dynamic light scattering measurements showed that Ca2+ at concentrations in the 0.5-2.0 mM range induced the aggregation of liposomes probably due to the formation of DPPS-enriched domains. However, annexin A5 avoided the aggregation of liposomes at Ca2+ concentrations lower than 1.0 mM. Surface pressure versus surface area isotherms showed that the adsorption of annexin A5 on the monolayers made of a mixture of DPPC and DPPS led to a reduction in the area of excess compared to the theoretical values, which confirmed that the protein favored attractive interactions among the membrane lipids. The stabilization of the lipid membranes by annexin A5 was also validated by recording the changes with time of the surface pressure. Finally, fluorescence microscopy images of lipid monolayers revealed the formation of spherical lipid-condensed domains that became unshaped and larger in the presence of annexin A5. Our data support the model that annexin A5 in matrix vesicles is recruited at the membrane sites enriched in phosphatidylserine and Ca2+ not only to contribute to the intraluminal mineral formation but also to stabilize the vesicles' membrane and prevent its premature rupture.

Nucleocapsid protein accumulates in renal tubular epithelium of a post-COVID-19 patient.

IMPORTANCE: Even though the coronavirus disease 2019 (COVID-19) pandemic is slowly developing into a conventional infectious disease, the long-term effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus infection are still not well understood. One of the problems is that many COVID-19 cases develop acute kidney injuries. Still, it is heavily debated whether SARS-CoV-2 virus enters and actively replicates in kidney tissue and if SARS-CoV-2 virus particles can be detected in kidney during or post-infection. Here, we demonstrated that nucleocapsid N protein was detected in kidney tubular epithelium of patients that already recovered form COVID-19. The presence of the abundantly produced N protein without signs of viral replication could have implications for the recurrence of kidney disease and have a continuing effect on the immune system.

Advancements in 3D spheroid imaging: Optimised cryosectioning and immunostaining techniques.

This article presents a modified protocol for embedding and sectioning spheroids and organoids, which are increasingly used in research due to their ability to emulate living tissue. The modifications aim to reduce the distortion and damage of these fragile structures during the embedding and sectioning process. The new method involves using optimized embedding containers, a modified embedding protocol, and optimized temperatures for cryosectioning. A heat-induced antigen retrieval protocol was tested and found to significantly increase immunostaining intensity without compromising spheroid integrity. The combined approach allowed for the creation of thinner cryosections, leading to clearer and more detailed images. The results suggest that the modified protocol could be widely adopted to enhance the imaging of spheroids and organoids.•Paraformaldehyde fixation of spheroids•Antigen retrieval treatment of spheroids•Embedding in freezing medium and cryosectioning.

Cardiomyocyte orientation recovery at micrometer scale reveals long-axis fiber continuum in heart walls.

Coordinated cardiomyocyte contraction drives the mammalian heart to beat and circulate blood. No consensus model of cardiomyocyte geometrical arrangement exists, due to the limited spatial resolution of whole heart imaging methods and the piecemeal nature of studies based on histological sections. By combining microscopy and computer vision, we produced the first-ever three-dimensional cardiomyocyte orientation reconstruction across mouse ventricular walls at the micrometer scale, representing a gain of three orders of magnitude in spatial resolution. We recovered a cardiomyocyte arrangement aligned to the long-axis direction of the outer ventricular walls. This cellular network lies in a thin shell and forms a continuum with longitudinally arranged cardiomyocytes in the inner walls, with a complex geometry at the apex. Our reconstruction methods can be applied at fine spatial scales to further understanding of heart wall electrical function and mechanics, and set the stage for the study of micron-scale fiber remodeling in heart disease.

First report of 'Candidatus Phytoplasma asteris' associated with yellowing, scorching and decline of almond trees in India.

The almond, a commercially important tree nut crop worldwide, is native to the Mediterranean region. Stone fruit trees are affected by at least 14 'Candidatus Phytoplasma' species globally, among which 'Candidatus Phytoplasma asteris' is one of the most widespread phytoplasma infecting Prunus dulcis, causing aster yellows disease. Recently, almond plantations of Nauni region were consistently affected by phytoplasma, as evidenced by visible symptoms, fluorescent microscopic studies and molecular characterization. During several surveys from May to September 2020-2022, almond aster yellows phytoplasma disease showing symptoms such as chlorosis, inward rolling, reddening, scorching and decline with an incidence as high as 40%. Leaf samples were collected from symptomatic almond trees and the presence of phytoplasma was confirmed through fluorescent microscopic studies by employing DAPI (4, 6-diamino-2-phenylindole) that showed distinctive light blue flourescent phytoplasma bodies in phloem sieve tube elements. The presence of phytoplasma in symptomatic almond trees was further confirmed using nested PCR with specific primer pairs followed by amplification of 16S rDNA and 16S-23S rDNA intergenic spacer (IS) fragments. Sequencing and BLAST analysis of expected amplicon of the 16S rDNA gene confirmed that the almond phytoplasma in Himachal Pradesh was identical to the aster yellows group phytoplasma. Phylogenetic analysis of 16S rDNA almond phytoplasma also grouped 'Prunus dulcis' aster yellows phytoplasma within 16SrI-B subgroup showed 94% nucleotide identity with 'Prunus dulcis' phytoplasma PAEs3 and 'Prunus dulcis' phytoplasma PAE28 from Iran. This research presents the first host report of 'Candidatus Phytoplasma asteris' infecting almonds in India, expanding the knowledge of the diversity and distribution of phytoplasma strains affecting almond trees globally.

Calix[4]arene-pyrazole conjugates as potential cancer therapeutics.

Tumor selectivity is yet a challenge in chemotherapy-based cancer treatment. A series of calixarenes derivatized at the lower rim with 3-phenyl-1H-pyrazole units with variable upper-rim substituent and conformations of macrocyclic core, alkyl chain length between heterocycle and core, as well as phenolic monomer (5-(4-tert-butylphenyloxy)methoxy-3-phenyl-1H-pyrazole) have been synthesized and characterized in a range of therapeutically relevant cellular models (M-HeLa, MCF7, A-549, PC3, Chang liver, and Wi38) from different target organs/systems. Specific cytotoxicity for M-HeLa cells has been observed in tert-butylcalix[4]arene pyrazoles in 1,3-alternate (compound 7b) and partial cone (compound 7c) conformations with low mutagenicity and haemotoxicity and in vivo toxicity in mice. Compounds 7b,c have induced mitochondrial pathway of apoptosis of M-HeLa cells through caspase-9 activation preceded by the cell cycle arrest at G0/G1 phase. A concomitant overexpression of DNA damage markers in pyrazole-treated M-HeLa cells suggests that calixarene pyrazoles target DNA, which was supported by the presence of interactions between calixarenes and ctDNA at the air-water interface.

Rapid microbial viability assay using scanning electron microscopy: a proof-of-concept using Phosphotungstic acid staining.

Multiple stains have been historically utilized in electron microscopy to provide proper contrast and superior image quality enabling the discovery of ultrastructures. However, the use of these stains in microbiological viability assessment has been limited. Phosphotungstic acid (PTA) staining is a common negative stain used in scanning electron microscopy (SEM). Here, we investigate the feasibility of a new SEM-PTA assay, aiming to determine both viable and dead microbes. The optimal sample preparation was established by staining bacteria with different PTA concentrations and incubation times. Once the assay conditions were set, we applied the protocol to various samples, evaluating bacterial viability under different conditions, and comparing SEM-PTA results to culture. The five minutes 10% PTA staining exhibited a strong distinction between viable micro-organisms perceived as hypo-dense, and dead micro-organisms displaying intense internal staining which was confirmed by high Tungsten (W) peak on the EDX spectra. SEM-PTA viability count after freezing, freeze-drying, or oxygen exposure, were concordant with culture. To our knowledge, this study is the first contribution towards PTA staining of live and dead bacteria. The SEM-PTA strategy demonstrated the feasibility of a rapid, cost-effective and efficient viability assay, presenting an open-view of the sample, and providing a potentially valuable tool for applications in microbiome investigations and antimicrobial susceptibility testing.