Real-Time Imaging of Calcium Dynamics in Human Sperm After Precise Single-Cell Stimulation.

Calcium signaling is a critical regulator of sperm activation and function during the processes of capacitation and fertilization. Here, we describe a combined method for calcium imaging of single, live human sperm in response to stimuli administered with a precisely targeted delivery technique. This protocol is an adaptation of techniques developed for studies of murine sperm [1, 2], and enables real-time monitoring of human sperm calcium dynamics with high spatiotemporal resolution and concurrent detection of acrosome exocytosis (AE), a functional endpoint of sperm capacitation and requirement for physiological fertilization.The described imaging technique provides a valuable tool for exploration of calcium regulation in human sperm, which is essential to answer important questions and knowledge gaps regarding the link between calcium dynamics, AE, and fertilization. The versatility of this technique can be amplified through use of various indicator dyes or integration with pharmacological strategies such as pre-treating sperm with inhibitors or activators targeting specific receptors, channels, or intracellular signaling pathways of interest. Beyond fundamental inquiries into sperm physiology, this method can also be applied to assess the impact of potential contraceptive compounds on calcium signaling, AE, and membrane integrity.

Study of the Mechanism of Perceived Rotational Acceleration of a Bionic Semicircular Canal on the Basis of the "Circular Geometry Hypothesis".

Academia often uses the "circular geometry hypothesis" to explain the sensing principle of the human semicircular canal (SCC) system for angular acceleration, which is widely accepted as an important angular acceleration sensor in the human balance system. On the basis of this hypothesis and the anatomical structure of human SCCs, a series of physical SCC models with different geometries at 4× magnification were prepared via three-dimensional printing and modification of hydrogels. Theoretical models of the SCC perception mechanism were established. Then, impulse angular acceleration, sinusoidal rotation, and sinusoidal linear stimulation were applied to the models, and their responses were visually observed and analyzed in detail. As a result, the circular SCC model had a larger system gain and a smaller phase difference for angular acceleration stimulation but a smaller system gain and a larger phase difference for linear acceleration stimulation. These results verified that the circular semicircular canal was more sensitive to angular acceleration. Our bionic model is hoped to be used for demonstrating the human SCC working process, facilitating researchers in better understanding of the working mechanism of the human SCC, or as a manual model for medical staff to simulate the diagnosis and treatment of human SCC.

Microglia are essential for tissue contraction in wound closure after brain injury in zebrafish larvae.

Wound closure after brain injury is crucial for tissue restoration but remains poorly understood at the tissue level. We investigated this process using in vivo observations of larval zebrafish brain injury. Our findings show that wound closure occurs within the first 24 h through global tissue contraction, as evidenced by live-imaging and drug inhibition studies. Microglia accumulate at the wound site before closure, and computational models suggest that their physical traction could drive this process. Depleting microglia genetically or pharmacologically impairs tissue repair. At the cellular level, live imaging reveals centripetal deformation of astrocytic processes contacted by migrating microglia. Laser severing of these contacts causes rapid retraction of microglial processes and slower retraction of astrocytic processes, indicating tension. Disrupting the lcp1 gene, which encodes the F-actin-stabilising protein L-plastin, in microglia results in failed wound closure. These findings support a mechanical role of microglia in wound contraction and suggest that targeting microglial mechanics could offer new strategies for treating traumatic brain injury.

Phenomenological Muscle Constitutive Model With Actin-Titin Binding for Simulating Active Stretching.

The force produced by a muscle depends on its contractile history, yet human movement simulations typically employ muscle models that define the force-length relationship from measurements of fiber force during isometric contractions. In these muscle models, the total force-length curve can have a negative slope at fiber lengths greater than the fiber length at which peak isometric force is produced. This region of negative stiffness can cause numerical instability in simulations. Experiments have found that the steady-state force in a muscle fiber following active stretching is greater than the force produced during a purely isometric contraction. This behavior is called residual force enhancement. We present a constitutive model that exhibits force enhancement, implemented as a hyperelastic material in the febio finite element software. There is no consensus on the mechanisms responsible for force enhancement; we adopt the assumption that the passive fiber force depends on the sarcomere length at the instant that the muscle is activated above a threshold. We demonstrate the numerical stability of our model using an eigenvalue analysis and by simulating a muscle whose fibers are of different lengths. We then use a three-dimensional muscle geometry to verify the effect of force enhancement on the development of stress and the distribution of fiber lengths. Our proposed muscle material model is one of the few models available that exhibits force enhancement and is suitable for simulations of active lengthening. We provide our implementation in febio so that others can reproduce and extend our results.

Physical Activity, Sedentary Behaviour, Sleep and Mental Wellbeing in Family Caregivers of Adults With Intellectual and/or Developmental Disabilities.

BACKGROUND: Canadian 24-h movement guidelines recommend that adults achieve 150 min per week of moderate-to-vigorous physical activity (MVPA), 7-9 h of sleep per night and spend no more than 8-h per day sedentary to optimise health and wellbeing. METHOD: Using a cross-sectional survey of 131 family caregivers of adults with intellectual and developmental disabilities, we aimed to (a) determine whether adherence to these guidelines predicts mental wellbeing in family caregivers and (b) explore the relationship between movement behaviours of family caregivers and their loved ones. RESULTS: While MVPA was found to weakly predict wellbeing, sleep and sedentary behaviour did not. The movement behaviours of the family caregivers were not closely related to that of their loved ones. CONCLUSIONS: Fostering physical activity is important to promote the wellbeing of adults with intellectual and developmental disabilities, as well as their family caregivers. Opportunities to be active together may be even more beneficial.

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.

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.

Inhibition mechanisms of perchlorate on the photosynthesis of cyanobacterium Synechocystis sp. PCC6803: Insights from physiology and transcriptome analysis.

Perchlorate (ClO4-) is a type of novel persistent inorganic pollutant that has gained increasing attention because of its high solubility, poor degradability, and widespread distribution. However, the impacts of perchlorate on aquatic autotrophs such cyanobacterium are still unclear. Herein, Synechocystis sp. PCC6803 (Synechocystis) was used to investigate the response mechanisms of perchlorate on cyanobacterium by integrating physiological and transcriptome analyses. Physiological results showed that perchlorate mainly damaged the photosystem of Synechocystis, and the inhibition degree of photosystem II (PSII) was severer than that of photosystem I (PSI). When the exposed cells were moved to a clean medium, the photosynthetic activities were slightly repaired but still lower than in the control group, indicating irreversible damage. Furthermore, perchlorate also destroyed the cellular ultrastructure and induced oxidative stress in Synechocystis. The antioxidant glutathione (GSH) content and the superoxide dismutase (SOD) enzyme activity were enhanced to scavenge harmful reactive oxygen (ROS) in Synechocystis. Transcriptome analysis revealed that the genes associated with "photosynthesis" and "electron transport" were significantly regulated. For instance, most genes related to PSI (e.g., psaf, psaJ) and the "electron transport chain" were upregulated, whereas most genes related to PSII (e.g., psbA3, psbD1, psbB, and psbC) were downregulated. Additionally, perchlorate also induced the expression of genes related to the antioxidant system (sod2, gpx, gst, katG, and gshB) to reduce oxidative damage. Overall, this study is the first to investigate the impacts and mechanisms of cyanobacterium under perchlorate stress, which is conducive to assessing the risk of perchlorate in aquatic environments.

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.

Recording of Age-Related Changes on Murine and Human Brain Slices.

Preparation of brain slices for electrophysiological and imaging experiments has been developed several decades ago, and the method is still widely used due to its simplicity and advantages over other techniques. It can be easily combined with other well established and recently developed methods as immunohistochemistry and morphological analysis or opto- and chemogenetics. Several aspects of this technique are covered by a plethora of excellent and detailed review papers, in which one can gain a deep insight of variations in it. In this chapter, I briefly describe the solutions, equipment, and preparation techniques routinely used in our laboratory. I also aim to present how certain "old school" brain slice lab devices can be made in a cost-efficient way. These devices can be easily adapted for the special needs of the experiments. I also aim to present some differences in the preparatory techniques of acutely isolated human brain tissue.

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.

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.

Characterization of Age-Dependent Changes in Skeletal Muscle Repair and Regeneration Using a Mouse Model of Acute Muscle Injury.

Acute skeletal muscle injury initiates a process of necrosis, debris clearance, and ultimately tissue regeneration via myogenesis. While skeletal muscle stem cells (MuSCs) are responsible for populating the proliferative myogenic progenitor pool to fuel muscle repair, recruited and resident immune cells have a central role in the regulation of muscle regeneration via the execution of phagocytosis and release of soluble factors that act directly on MuSCs to regulate myogenic differentiation. Therefore, the timing of MuSC proliferation and differentiation is closely linked to the populations and behaviors of immune cells present within skeletal muscle. This has important implications for aging and muscle repair, as systemic changes in immune system function contribute to a decline in muscle regenerative capacity. Here, we present adapted protocols for the isolation of mononuclear cells from skeletal muscles for the quantification of immune cell populations using flow cytometry. We also describe a cardiotoxin skeletal muscle injury protocol and detail the expected outcomes including immune cell infiltration to the injured sites and formation of new myocytes. As immune cell function is substantially influenced by aging, we extend these approaches and outcomes to aged mice.

Impact of pre-exposure stress on the growth and viability of Lactobacillus acidophilus in regular, buriti pulp and orange byproduct fermented milk products.

The loss of probiotics viability in yogurts and fermented milk is a significant challenge in producing yogurt and fermented milk. Thus, pre-exposure of probiotics to stress conditions can be a viable alternative to increase the probiotic viability. Moreover, the use of fruit pulp and agro-industrial residues in these products has demonstrated promising results in promoting growth and improving the viability of probiotics. Thus, this study aimed to evaluate the effects of pre-exposure to acid, oxidative and osmotic stress on the growth and viability of Lactobacillus acidophilus in yogurts and naturally fermented milk containing buriti (Mauritia flexuosa Mart.) pulp or orange byproduct. L. acidophilus was individually pre-exposed to acid, oxidative, and osmotic stress and used in the production of yogurts and fermented milk to determine both the acidification profile and growth of the cultures. Furthermore, during cold storage, the post-acidification profiles and viability of microbial cultures added to the yogurts and fermented milk were monitored. Results showed that pre-exposure to stress conditions influenced the growth parameters as the growth rate (µ) and lag phase (λ) of L. acidophilus and the starter cultures of S. thermophilus and L. delbrueckii subsp. bulgaricus. Moreover, an increase in the viability of L. acidophilus - pre-exposed to acid stress - was observed on the 21st day of storage of natural yogurts containing orange byproduct compared with non-stressful conditions. This study reports new data on the growth of probiotic cultures pre-exposed to stress conditions in products added of pulps and agro-industrial residues, which have not yet been shown in the literature.

Development and application of a bacteriophage cocktail for Shigella flexneri biofilm inhibition on the stainless steel surface.

Food contamination and biofilm formation by Shigella in food processing facilities are major causes of acute gastrointestinal infection and mortality in humans. Bacteriophages (phages) are promising alternatives to antibiotics in controlling plankton and biofilms in food matrices. This study isolated two novel phages, S2_01 and S2_02, with lytic activity against various Shigella spp. From sewage samples. Transmission electron microscopy revealed that phages S2_01 and S2_02 belonged to the Caudovirales order. On characterizing their lytic ability, phage S2_01 initially exhibited relatively weak antibacterial activity, while phage S2_02 initially displayed rapid antibacterial activity after phage application. A combination of these phages in a 1:9 ratio was selected, as it has been suggested to elicit the most rapid and sustained lysis ability for up to 24 h. It demonstrated lytic activity against various foodborne pathogens, including six Shigella spp. The phage cocktail exhibited biofilm inhibition and disruption abilities of approximately 79.29% and 42.55%, respectively, after 24 h in a 96-well microplate. In addition, inhibition (up to 23.42%) and disruption (up to 19.89%) abilities were also observed on stainless steel surfaces, and plankton growth was also significantly suppressed. Therefore, the phage cocktail formulated in this study displays great potential as a biological control agent in improving food safety against biofilms and plankton.

SARS-CoV-2 surrogate bacteriophage φ6 cross-contamination between fruits and gloves, survival on discarded gloves and inactivation by photodynamic treatment.

This study assessed the SARS-CoV-2 surrogate bacteriophage φ6 cross-contamination between high-density polyethylene or polyvinyl chloride gloves and fruits (tomato and cucumber) using different inoculum levels (6.0 and 4.0 log PFU/sample). Bacteriophage φ6 survival on contaminated gloves was assessed over 9 days at 25 °C. The effectiveness of photodynamic treatment using curcumin as a photosensitizer to inactivate φ6 on fruits was determined. The fruit type and the glove material influenced the φ6 transfer. Longer contact times resulted in greater φ6 transfer. The highest φ6 transfer occurred from tomato to HDPE glove (0.8% or -1.1 log % transfer) after 30 s of contact at the higher inoculum level. Bacteriophage φ6 was detected on cross-contaminated HDPE gloves for up to 6 days. Bacteriophage φ6 survived better on vinyl gloves cross-contaminated by cucumber vs. tomato (detected up to 6 vs 3 days). Photodynamic inactivation of φ6 was time-dependent and varied with the tested fruit but was not influenced by viral starting concentration. Photodynamic treatment decreased the φ6 titer by 3.0 and 2.2 log PFU/sample in tomato and cucumber, respectively. Transmission electronic microscopy showed that photodynamic treatment changed the structure of the φ6 capsid. These findings may help in the management of SARS-CoV-2 contamination risks in fruit handling. They may also help in the establishment of effective measures to manage cross-contamination risk.

Commercial bacteriophage preparations for the control of Listeria monocytogenes and Shiga toxin-producing Escherichia coli in raw and pasteurized milk.

Listeria monocytogenes was the etiologic agent in nearly all recent outbreaks in North America attributed to pasteurized dairy products, whereas Escherichia coli O157 infections were responsible for most of the rare, yet serious complications from outbreaks involving unpasteurized dairy. This study determined the susceptibility of selected strains of L. monocytogenes and Shiga toxin-producing E. coli (STEC) to commercial phage preparations and their ability to control these pathogens in pasteurized and raw milk during 7-day storage at 7 °C. Both phage products demonstrated high lytic efficiency against 17 strains of L. monocytogenes whereas the efficiency of E. coli phages was more variable against 11 strains of O157 and non-O157 STEC. Broth microdilution assays identified effective endpoint multiplicities of infection (MOI) ranging from log 2.53 to 5.13, which differed between strains of L. monocytogenes and phage products. Mean log MOIs of E. coli phages against STEC also varied within and between products from 0.43 to 7.05. Despite these observations, the change in counts over time of three L. monocytogenes strains exposed to phage in pasteurized milk (log MOI 6) was similar with counts âˆ¼ 4 log CFU/mL lower than control at day 7. Results for STEC O157 varied by strain but counts were lower than control in all cases by 72 h thorough day 7. Titers on isolates of both pathogens isolated from pasteurized milk indicated that the surviving populations were less susceptible to phage. The addition of a phage preparation to raw milk did not reduce populations of either pathogen or affect the change in counts of any strain over time when compared to control. Reduced efficacy in raw milk may be attributed to reduced phage binding as titers in raw milk decreased steadily (∼2 log PFU/mL) during storage. Commercial phage products may be a promising pathogen control intervention for pasteurized dairy products, warranting further investigation.