Isolation and Characterization of Extracellular Vesicles to Activate Retina Regeneration.

Mammals do not possess the ability to spontaneously repair or regenerate damaged retinal tissue. In contrast to teleost fish which are capable of retina regeneration through the action of Müller glia, mammals undergo a process of reactive gliosis and scarring that inhibits replacement of lost neurons. Thus, it is important to discover novel methods for stimulating mammalian Müller glia to dedifferentiate and produce progenitor cells that can replace lost retinal neurons. Inducing an endogenous regenerative pathway mediated by Müller glia would provide an attractive alternative to stem cell injections or gene therapy approaches. Extracellular vesicles (EVs) are now recognized to serve as a novel form of cell-cell communication through the transfer of cargo from donor to recipient cells or by the activation of signaling cascades in recipient cells. EVs have been shown to promote proliferation and regeneration raising the possibility that delivery of EVs could be a viable treatment for visual disorders. Here, we provide protocols to isolate EVs for use in retina regeneration experiments.

Automated In Vivo Phenotypic Screening Platform for Identifying Factors that Affect Cell Regeneration Kinetics.

Various strategies for replacing retinal neurons lost in degenerative diseases are under investigation, including stimulating the endogenous regenerative capacity of Müller Glia (MG) as injury-inducible retinal stem cells. Inherently regenerative species, such as zebrafish, have provided key insights into mechanisms regulating MG dedifferentiation to a stem-like state and the proliferation of MG and MG-derived progenitor cells (MGPCs). Interestingly, promoting MG/MGPC proliferation is not sufficient for regeneration, yet mechanistic studies are often focused on this measure. To fully account for the regenerative process, and facilitate screens for factors regulating cell regeneration, an assay for quantifying cell replacement is required. Accordingly, we adapted an automated reporter-assisted phenotypic screening platform to quantify the pace of cellular regeneration kinetics following selective cell ablation in larval zebrafish. Here, we detail a method for using this approach to identify chemicals and genes that control the rate of retinal cell regeneration following selective retinal cell ablation.

Assessment of Viability of Listeria monocytogenes by Flow Cytometry.

In food industry, Listeria monocytogenes contamination can occur accidentally despite the quality control of raw materials and factory. Decontamination processes or inhibitory effects of ingredients/additives in food products are set up to ensure compliance with hygiene and microbiological criteria. These actions represent stresses for the pathogenic agent, causing fluctuations in its physiological states. Moreover, during these environmental stresses, Listeria monocytogenes can enter in a viable but nonculturable (VBNC) state which is not detected by plate counting but by flow cytometry. This technique coupled with cell staining by fluorescent dyes offers the possibility to assess different physiological states based on different cellular parameters: enzymatic activity, transmembrane integrity, membrane potential, and respiratory activity. In this chapter, we present a method to assess the viability of foodborne pathogens using a double-staining principle based on the assessment of membrane integrity and intracellular esterase activity.

Comparing Gene Expression Between Planktonic and Biofilm Cells of Foodborne Bacterial Pathogens Through RT-qPCR.

Like most microorganisms, important foodborne pathogenic bacteria, such as Salmonella enterica, Listeria monocytogenes, and several others as well, can attach to surfaces, of either abiotic or biotic nature, and create biofilms on them, provided the existence of supportive environmental conditions (e.g., permissive growth temperature, adequate humidity, and nutrient presence). Inside those sessile communities, the enclosed bacteria typically present a gene expression profile that differs from the one that would be displayed by the same cells growing planktonically in liquid media (free-swimming cells). This altered gene expression has important consequences on cellular physiology and behavior, including stress tolerance and induction of virulence. In this chapter, the methodology to use reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to monitor and comparatively quantify expression changes in preselected genes of bacteria between planktonic and biofilm growth modes is presented.

Preventive and therapeutic effects of a super-multivalent sialylated filamentous bacteriophage against the influenza virus.

The resurgence of influenza viruses as a significant global threat emphasizes the urgent need for innovative antiviral strategies beyond existing treatments. Here, we present the development and evaluation of a novel super-multivalent sialyllactosylated filamentous phage, termed t-6SLPhage, as a potent entry blocker for influenza A viruses. Structural variations in sialyllactosyl ligands, including linkage type, valency, net charge, and spacer length, were systematically explored to identify optimal binding characteristics against target hemagglutinins and influenza viruses. The selected SLPhage equipped with optimal ligands, exhibited exceptional inhibitory potency in in vitro infection inhibition assays. Furthermore, in vivo studies demonstrated its efficacy as both a preventive and therapeutic intervention, even when administered post-exposure at 2 days post-infection, under 4 lethal dose 50% conditions. Remarkably, co-administration with oseltamivir revealed a synergistic effect, suggesting potential combination therapies to enhance efficacy and mitigate resistance. Our findings highlight the efficacy and safety of sialylated filamentous bacteriophages as promising influenza inhibitors. Moreover, the versatility of M13 phages for surface modifications offers avenues for further engineering to enhance therapeutic and preventive performance.

Gradient conducting polymer surfaces with netrin-1-conjugation promote axon guidance and neuron transmission of human iPSC-derived retinal ganglion cells.

Major advances have been made in utilizing human-induced pluripotent stem cells (hiPSCs) for regenerative medicine. Nevertheless, the delivery and integration of hiPSCs into target tissues remain significant challenges, particularly in the context of retinal ganglion cell (RGC) restoration. In this study, we introduce a promising avenue for providing directional guidance to regenerated cells in the retina. First, we developed a technique for construction of gradient interfaces based on functionalized conductive polymers, which could be applied with various functionalized ehthylenedioxythiophene (EDOT) monomers. Using a tree-shaped channel encapsulated with a thin PDMS and a specially designed electrochemical chamber, gradient flow generation could be converted into a functionalized-PEDOT gradient film by cyclic voltammetry. The characteristics of the successfully fabricated gradient flow and surface were analyzed using fluorescent labels, time of flight secondary ion mass spectrometry (TOF-SIMS), and X-ray photoelectron spectroscopy (XPS). Remarkably, hiPSC-RGCs seeded on PEDOT exhibited improvements in neurite outgrowth, axon guidance and neuronal electrophysiology measurements. These results suggest that our novel gradient PEDOT may be used with hiPSC-based technologies as a potential biomedical engineering scaffold for functional restoration of RGCs in retinal degenerative diseases and optic neuropathies.

Application of electroencephalogram (EEG) in the study of the influence of different contents of alcohol and Baijiu on brain perception.

Alcoholic beverages flavour is complex and unique with different alcohol content, and the application of flavour perception could improve the objectivity of flavour evaluation. This study utilized electroencephalogram (EEG) to assess brain reactions to alcohol percentages (5 %-53 %) and Baijiu's complex flavours. The findings demonstrate the brain's proficiency in discerning between alcohol concentrations, evidenced by increasing physiological signal strength in tandem with alcohol content. When contrasted with alcohol solutions of equivalent concentrations, Baijiu prompts a more significant activation of brain signals, underscoring EEG's capability to detect subtleties due to flavour complexity. Additionally, the study reveals notable correlations, with δ and α wave intensities escalating in response to alcohol stimulation, coupled with substantial activation in the frontal, parietal, and right temporal regions. These insights verify the efficacy of EEG in charting the brain's engagement with alcoholic flavours, setting the stage for more detailed exploration into the neural encoding of these sensory experiences.

2D Janus carrier-enabled trojan horse: Gallium delivery for the sequential therapy of biofilm associated infection.

Biofilm-associated infections (BAIs) continue to pose a major challenge in the medical field. Nanomedicine, in particular, promises significant advances in combating BAIs through the introduction of a variety of nanomaterials and nano-antimicrobial strategies. However, studies to date have primarily focused on the removal of the bacterial biofilm and neglect the subsequent post-biofilm therapeutic measures for BAIs, rendering pure anti-biofilm strategies insufficient for the holistic recovery of affected patients. Herein, we construct an emerging dual-functional composite nanosheet (SiHx@Ga) that responds to pHs fluctuation in the biofilm microenvironment to enable a sequential therapy of BAIs. In the acidic environment of biofilm, SiHx@Ga employs the self-sensitized photothermal Trojan horse strategy to effectively impair the reactive oxygen species (ROS) defense system while triggering oxidative stress and lipid peroxidation of bacteria, engendering potent antibacterial and anti-biofilm effects. Surprisingly, in the post-treatment phase, SiHx@Ga adsorbs free pathogenic nucleic acids released after biofilm destruction, generates hydrogen with ROS-scavenging and promotes macrophage polarization to the M2 type, effectively mitigating damaging inflammatory burst and promoting tissue healing. This well-orchestrated strategy provides a sequential therapy of BAIs by utilizing microenvironmental variations, offering a conceptual paradigm shift in the field of nanomedicine anti-infectives.

Activation of peroxymonosulfate by sustainable biomass-based carbon nanotubes for controlling the spread of plant viruses in water environments.

With the increasing demand for water in hydroponic systems and agricultural irrigation, viral diseases have seriously affected the yield and quality of crops. By removing plant viruses in water environments, virus transmission can be prevented and agricultural production and ecosystems can be protected. But so far, there have been few reports on the removal of plant viruses in water environments. Herein, in this study, easily recyclable biomass-based carbon nanotubes catalysts were synthesized with varying metal activities to activate peroxymonosulfate (PMS). Among them, the magnetic 0.125Fe@NCNTs-1/PMS system showed the best overall removal performance against pepper mild mottle virus, with a 5.9 log10 removal within 1 min. Notably, the key reactive species in the 0.125Fe@NCNTs-1/PMS system is 1O2, which can maintain good removal effect in real water matrices (river water and tap water). Through RNA fragment analyses and label free analysis, it was found that this system could effectively cleave virus particles, destroy viral proteins and expose their genome. The capsid protein of pepper mild mottle virus was effectively decomposed where serine may be the main attacking sites by 1O2. Long viral RNA fragments (3349 and 1642 nt) were cut into smaller fragments (∼160 nt) and caused their degradation. In summary, this study contributes to controlling the spread of plant viruses in real water environment, which will potentially help protect agricultural production and food safety, and improve the health and sustainability of ecosystems.

Combined biochar and wheat-derived endophytic bacteria reduces cadmium uptake in wheat grains in a metal-polluted soil.

In this study, two wheat-derived cadmium (Cd)-immobilizing endophytic Pseudomonas paralactis M14 and Priestia megaterium R27 were evaluated for their effects on wheat tissue Cd uptake under hydroponic conditions. Then, the impacts of the biochar (BC), M14+R27 (MR), and BC+MR treatments on wheat Cd uptake and the mechanisms involved were investigated at the jointing, heading, and mature stages of wheat plants under field-plot conditions. A hydroponic experiment showed that the MR treatment significantly decreased the above-ground tissue Cd content compared with the M14 or R27 treatment. The BC+MR treatment reduced the grain Cd content by 51.5%-67.7% and Cd translocation factor at the mature stage of wheat plants and increased the organic matter-bound Cd content by 31%-75% in the rhizosphere soils compared with the BC or MR treatment. Compared with the BC or MR treatment, the relative abundances of the biomarkers associated with Gemmatimonas, Altererythrobacter, Gammaproteobacteria, Xanthomonadaceae, Phenylobacterium, and Nocardioides in the BC+MR-treated rhizosphere microbiome decreased and negatively correlated with the organic matter-bound Cd contents. In the BC+MR-treated root interior microbiome, the relative abundance of the biomarker belonging to Exiguobacterium increased and negatively correlated with the Cd translocation factor, while the relative abundance of the biomarker belonging to Pseudonocardiaceae decreased and positively correlated with the Cd translocation factor. Our findings suggested that the BC+MR treatment reduced Cd availability and Cd transfer through affecting the abundances of these specific biomarkers in the rhizosphere soil and root interior microbiomes, leading to decreased wheat grain Cd uptake in the contaminated soil.

4-Nitrophenol at environmentally relevant concentrations mediates reproductive toxicity in Caenorhabditis elegans via metabolic disorders-induced estrogen signaling pathway.

4-Nitrophenol (4-NP), as a toxic and refractory pollutant, has generated significant concern due to its adverse effects. However, the potential toxic effects and mechanism remained unclear. In this study, the reproduction, development, locomotion and reactive oxygen species (ROS) production of Caenorhabditis elegans were investigated to evaluate the 4-NP toxicity. We used metabolomics to assess the potential damage mechanisms. The role of metabolites in mediating the relationship between 4-NP and phenotypes was examined by correlation and mediation analysis. 4-NP (8 ng/L and 8 µg/L) caused significant reduction of brood size, ovulation rate, total germ cells numbers, head thrashes and body bends, and an increase in ROS. However, the oosperm numbers in uterus, body length and body width were decreased in 8 µg/L. Moreover, 36 differential metabolites were enriched in the significant metabolic pathways, including lysine biosynthesis, ß-alanine metabolism, tryptophan metabolism, pentose phosphate pathway, pentose and glucuronate interconversions, amino sugar and nucleotide sugar metabolism, starch and sucrose metabolism, galactose metabolism, propanoate metabolism, glycerolipid metabolism, and estrogen signaling pathway. The mechanism of 4-NP toxicity was that oxidative stress caused by the perturbation of amino acid, which had effects on energy metabolism through disturbing carbohydrate and lipid metabolism, and finally affected the estrogen signaling pathway to exert toxic effects. Moreover, correlation and mediation analysis showed glycerol-3P, glucosamine-6P, glucosamine-1P, UDP-galactose, L-aspartic acid, and uracil were potential markers for the reproduction and glucose-1,6P2 for developmental toxicity. The results provided insight into the pathways involved in the toxic effects caused by 4-NP and developed potential biomarkers to evaluate 4-NP toxicity.

The dynamic reconfiguration of the functional network during episodic memory task predicts the memory performance.

Episodic memory is essential for forming and retaining personal experiences, representing a fundamental aspect of human cognition. Traditional studies of episodic memory have typically used static analysis methods, viewing the brain as an unchanging entity and overlooking its dynamic properties over time. In this study, we utilized dynamic functional connectivity analysis on fMRI data from healthy adults performing an episodic memory task. We quantified integration and recruitment metrics and examined their correlation with memory performance using Pearson correlation. During encoding, integration across the entire brain, especially within the frontoparietal subnetwork, was significantly correlated with memory performance. During retrieval, recruitment becomes significantly associated with memory performance in visual subnetwork, somatomotor subnetwork, and ventral attention subnetwork. At the nodal level, a significant negative correlation was observed between memory scores and integration of the anterior cingulate gyrus, precentral gyrus, and inferior frontal gyrus within the frontoparietal network during encoding task. During retrieval task, a significant negative correlation was found between memory scores and recruitment in the left progranular cortex and right transverse gyral ventral, whereas positive correlations were seen in the right posterior inferior temporal, left middle temporal, right frontal operculum, and left operculum nodes. Moreover, the dynamic reconfiguration of the functional network was predictive of predict memory performance, as demonstrated by a significant correlation between actual and predicted memory scores. These findings advance our understanding network mechanisms underlying memory processes and developing intervention approaches for memory-related disorders as they shed light on critical factors involved in cognitive processes and provide a deeper understanding of the underlying mechanisms driving cognitive function.

A systems view of the vascular endothelium in health and disease.

The dysfunction of blood-vessel-lining endothelial cells is a major cause of mortality. Although endothelial cells, being present in all organs as a single-cell layer, are often conceived as a rather inert cell population, the vascular endothelium as a whole should be considered a highly dynamic and interactive systemically disseminated organ. We present here a holistic view of the field of vascular research and review the diverse functions of blood-vessel-lining endothelial cells during the life cycle of the vasculature, namely responsive and relaying functions of the vascular endothelium and the responsive roles as instructive gatekeepers of organ function. Emerging translational perspectives in regenerative medicine, preventive medicine, and aging research are developed. Collectively, this review is aimed at promoting disciplinary coherence in the field of angioscience for a broader appreciation of the importance of the vasculature for organ function, systemic health, and healthy aging.

Movements of the pelvic bones of expectant mothers during vaginal delivery.

Subtle but demonstrable movements in the expectant mother's pelvis occur during vaginal delivery in all the pelvic joints and anatomical planes of the body (sagittal, frontal, and transverse). The purpose of these movements is to gradually expand the space in the lesser pelvis via widening of the individual pelvic planes so that the newborn's head can enter the pelvic inlet, safely pass through the narrow planes of the pelvis, and through the pelvic outlet. From the point of view of biomechanics, these movements are described in literature as counternutation and nutation of the sacrum and iliac bone. The counternutation of the sacrum helps to expand the plane of the pelvic inlet. The nutation of the sacrum assists in expanding the plane of the pelvic width, height, and outlet. These physiological movements are affected by the body constitution, the state of the myofascial and skeletal systems of the mother, and furthermore, by hormonal disjunction of the connections in the expectant mother's pelvis together with the progress of the delivery mechanism itself. The main factor that determines the range of movement in the individual joints, and therefore adequate expansion of the individual pelvic planes, is the position of the mother during delivery. Engagement of active movements of the mother together with application of passive stretching of the soft tissues in the lower lumbar area and in the hip joints are both needed for maximum expansion of the individual pelvic planes and utilization of the maximum useful capacity of the mother's pelvis during delivery. These movements help invoke the abduction forces on muscles, tendons, and ligaments in the pelvis that lead to the optimum setting of the joints during which delivery movements happen. The specific movements in the pelvic joints predetermine whether nutation or counternutation is possible, and therefore if the newborn's head can progress to the pelvic inlet or pass through the narrow and wide pelvic planes, and the pelvic outlet. The knowledge of these biomechanical principles and movements in the pelvis during delivery enables obstetricians and midwives to understand how the movements in the hip joints of the expectant mother can positively impact the spatial ratios in the lesser pelvis, and how to support further progress in the event of non-progressive labour.

Is it really over when it is over? physical, mental and emotional health status of long-term breast cancer survivors compared to healthy matched controls.

PURPOSE: This study aimed to assess pain, fitness condition, physical activity (PA) level, comorbidities, cancer-related fatigue (CRF), mood state and health-related quality of life (HRQoL) in long-term breast cancer survivors (LTBCS) compared to women without cancer history, matched by age, weight, height, and educational level. METHODS: A cross-sectional study conducted in Granada between April 2018 and July 2023 involved 80 LTBCS and 80 matched controls. Pain, fitness condition, PA level, comorbidities, CRF, mood state, and HRQoL were evaluated ≥ 5 years post-diagnosis using validated instruments. RESULTS: LTBCS, compared to the controls, reported significantly higher levels of "pain intensity and interference", CRF (in all domains and > 40% exhibited moderate-to-severe fatigue levels), "sadness-depression", "anxiety", "anger/hostility", and "symptom scales" (All: P = .000 to .027). Moreover, 66.25% of LTBCS not only did not reach recommended PA levels (P = .035), but also presented significantly lower levels of "general physical fitness", "muscular strength", "happiness", "functioning scales" (except "emotional functioning"), and "global health status" (All: P = .000 to .048). CONCLUSION: LTBCS still suffer from physical (pain, fitness condition, and CRF), both mental and emotional (sadness-depression, anxiety and anger/hostility) long-term side effects as well as multiple HRQoL issues (including lower levels of physical functioning and higher levels of symptoms). These findings highlight the chronic nature of this disease and the importance of continuing long- term follow-up care for survivors many years after the diagnosis of breast cancer.

Communicating Across Cell Walls: Structure, Evolution, and Regulation of Plasmodesmatal Transport in Plants.

Plasmodesmata are conduits in plant cell walls that allow neighboring cells to communicate and exchange resources. Despite their central importance to plant development and physiology, our understanding of plasmodesmata is relatively limited compared to other subcellular structures. In recent years, technical advances in electron microscopy, mass spectrometry, and phylogenomics have illuminated the structure, composition, and evolution of plasmodesmata in diverse plant lineages. In parallel, forward genetic screens have revealed key signaling pathways that converge to regulate plasmodesmatal transport, including chloroplast-derived retrograde signaling, phytohormone signaling, and metabolic regulation by the conserved eukaryotic Target of Rapamycin kinase. This review summarizes our current knowledge of the structure, evolution, and regulation of plasmodesmatal transport in plants.

Oocyte Health and Quality: Implication of Mitochondria-related Organelle Interactions.

Among factors like hormonal imbalance and uterine condition, oocyte quality is regarded as one of the key factors involved in age-related decline in the reproductive capacity. Here, are discussions about the functions played by organelles within the oocyte in forming the next generation that is more suitable for survival. Many insights on the adaptation to aging and maintenance of quality can be obtained from: interactions between mitochondria and other organelles that enable the long life of primordial oocytes; characteristics of organelle interactions after breaking dormancy from primary oocytes to mature oocytes; and characteristics of interactions between mitochondria and other organelles of aged oocytes collected during the ovulatory cycle from elderly individuals and animals. This information would potentially be beneficial to the development of future therapeutic methods or agents.

Airineme-Mediated Intercellular Communication.

Intercellular communication is indispensable across multicellular organisms, and any aberration in this process can give rise to significant anomalies in developmental and homeostatic processes. Thus, a comprehensive understanding of its mechanisms is imperative for addressing human health-related concerns. Recent advances have expanded our understanding of intercellular communication by elucidating additional signaling modalities alongside established mechanisms. Notably, cellular protrusion-mediated long-range communication, characterized by physical contact through thin and elongated cellular protrusions between cells involved in signal transmission and reception, has emerged as a significant intercellular signaling paradigm. This chapter delves into the exploration of a signaling cellular protrusion termed 'airinemes,' discovered in the zebrafish skin. It covers their identified signaling roles and the cellular and molecular mechanisms that underpin their functionality.

Organelle Interactions in Plant Cells.

The sequestration of enzymes and associated processes into sub-cellular domains, called organelles, is considered a defining feature of eukaryotic cells. However, what leads to specific outcomes and allows a eukaryotic cell to function singularly is the interactivity and exchanges between discrete organelles. Our ability to observe and assess sub-cellular interactions in living plant cells has expanded greatly following the creation of fluorescent fusion proteins targeted to different organelles. Notably, organelle interactivity changes quickly in response to stress and reverts to a normal less interactive state as homeostasis is re-established. Using key observations of some of the organelles present in a plant cell, this chapter provides a brief overview of our present understanding of organelle interactions in plant cells.

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.