Germinal Centers.

Germinal centers (GCs) are microanatomical sites of B cell clonal expansion and antibody affinity maturation. Therein, B cells undergo the Darwinian process of somatic diversification and affinity-driven selection of immunoglobulins that produces the high-affinity antibodies essential for effective humoral immunity. Here, we review recent developments in the field of GC biology, primarily as it pertains to GCs induced by infection or immunization. First, we summarize the phenotype and function of the different cell types that compose the GC, focusing on GC B cells. Then, we review the cellular and molecular bases of affinity-dependent selection within the GC and the export of memory and plasma cells. Finally, we present an overview of the emerging field of GC clonal dynamics, focusing on how GC and post-GC selection shapes the diversity of antibodies secreted into serum.

Light-Sheet Microscopy and Its Potential for Understanding Developmental Processes.

The ability to visualize and quantitatively measure dynamic biological processes in vivo and at high spatiotemporal resolution is of fundamental importance to experimental investigations in developmental biology. Light-sheet microscopy is particularly well suited to providing such data, since it offers exceptionally high imaging speed and good spatial resolution while minimizing light-induced damage to the specimen. We review core principles and recent advances in light-sheet microscopy, with a focus on concepts and implementations relevant for applications in developmental biology. We discuss how light-sheet microcopy has helped advance our understanding of developmental processes from single-molecule to whole-organism studies, assess the potential for synergies with other state-of-the-art technologies, and introduce methods for computational image and data analysis. Finally, we explore the future trajectory of light-sheet microscopy, discuss key efforts to disseminate new light-sheet technology, and identify exciting opportunities for further advances.

Coherent light scattering from cellular dynamics in living tissues.

This review examines the biological physics of intracellular transport probed by the coherent optics of dynamic light scattering from optically thick living tissues. Cells and their constituents are in constant motion, composed of a broad range of speeds spanning many orders of magnitude that reflect the wide array of functions and mechanisms that maintain cellular health. From the organelle scale of tens of nanometers and upward in size, the motion inside living tissue is actively driven rather than thermal, propelled by the hydrolysis of bioenergetic molecules and the forces of molecular motors. Active transport can mimic the random walks of thermal Brownian motion, but mean-squared displacements are far from thermal equilibrium and can display anomalous diffusion through Lévy or fractional Brownian walks. Despite the average isotropic three-dimensional environment of cells and tissues, active cellular or intracellular transport of single light-scattering objects is often pseudo-one-dimensional, for instance as organelle displacement persists along cytoskeletal tracks or as membranes displace along the normal to cell surfaces, albeit isotropically oriented in three dimensions. Coherent light scattering is a natural tool to characterize such tissue dynamics because persistent directed transport induces Doppler shifts in the scattered light. The many frequency-shifted partial waves from the complex and dynamic media interfere to produce dynamic speckle that reveals tissue-scale processes through speckle contrast imaging and fluctuation spectroscopy. Low-coherence interferometry, dynamic optical coherence tomography, diffusing-wave spectroscopy, diffuse-correlation spectroscopy, differential dynamic microscopy and digital holography offer coherent detection methods that shed light on intracellular processes. In health-care applications, altered states of cellular health and disease display altered cellular motions that imprint on the statistical fluctuations of the scattered light. For instance, the efficacy of medical therapeutics can be monitored by measuring the changes they induce in the Doppler spectra of livingex vivocancer biopsies.

A mathematical model for wound healing in the reef-building coral Pocillopora damicornis.

Coral reefs, among the most diverse ecosystems on Earth, currently face major threats from pollution, unsustainable fishing practices , and perturbations in environmental parameters brought on by climate change. Corals also sustain regular wounding from other sea life and human activity. Recent reef restoration practices have even involved intentional wounding by systematically breaking coral fragments and relocating them to revitalize damaged reefs, a practice known as microfragmentation. Despite its importance, very little research has explored the inner mechanisms of wound healing in corals. Some reef-building corals have been observed to initiate an immunological response to wounding similar to that observed in mammalian species. Utilizing prior models of wound healing in mammalian species as the mathematical basis, we formulated a mechanistic model of wound healing, including observations of the immune response and tissue repair in scleractinian corals for the species Pocillopora damicornis. The model consists of four differential equations which track changes in remaining wound debris, number of cells involved in inflammation, number of cells involved in proliferation, and amount of wound closure through re-epithelialization. The model is fit to experimental wound size data from linear and circular shaped wounds on a live coral fragment. Mathematical methods, including numerical simulations and local sensitivity analysis, were used to analyze the resulting model. The parameter space was also explored to investigate drivers of other possible wound outcomes. This model serves as a first step in generating mathematical models for wound healing in corals that will not only aid in the understanding of wound healing as a whole, but also help optimize reef restoration practices and predict recovery behavior after major wounding events.

Characterizing Chemical, Environmental, and Stimulated Subcellular Physical Characteristics of Size-Fractionated PMs Down to PM0.1.

Air pollution, especially particulate matter (PM), is a significant environmental pollution worldwide. Studying the chemical, environmental, and life-related cellular physical characteristics of size-fractionated PMs is important because of their different degrees of harmful effects on human respiratory tracts and organ systems, causing severe diseases. This study evaluates the chemical components of size-fractionated PMs down to PM0.1 collected during a biomass-burning episode, including elemental/organic carbon and trace elements. Single particle sizes and distributions of PM0.1, PM0.5-0.1, PM1.0-0.5, and PM2.5-1.0 were analyzed by scanning electron microscopy and Zeta sizer. Two commonly used cell lines, e.g., HeLa and Cos7 cells, and two respiratory-related cell lines including lung cancer/normal cells were utilized for cell cytotoxicity experiments, revealing the key effects of particle sizes and concentrations. A high-speed scanning ion conductance microscope explored particle-stimulated subcellular physical characteristics for all cell lines in dynamics, including surface roughness (SR) and elastic modulus (E). The statistical results of SR showed distinct features among different particle sizes and cell types while a E reduction was universally found. This work provides a comprehensive understanding of the chemical, environmental, and cellular physical characteristics of size-fractionated PMs and sheds light on the necessity of controlling small-sized PM exposures.

Biosensor-Enhanced Organ-on-a-Chip Models for Investigating Glioblastoma Tumor Microenvironment Dynamics.

Glioblastoma, an aggressive primary brain tumor, poses a significant challenge owing to its dynamic and intricate tumor microenvironment. This review investigates the innovative integration of biosensor-enhanced organ-on-a-chip (OOC) models as a novel strategy for an in-depth exploration of glioblastoma tumor microenvironment dynamics. In recent years, the transformative approach of incorporating biosensors into OOC platforms has enabled real-time monitoring and analysis of cellular behaviors within a controlled microenvironment. Conventional in vitro and in vivo models exhibit inherent limitations in accurately replicating the complex nature of glioblastoma progression. This review addresses the existing research gap by pioneering the integration of biosensor-enhanced OOC models, providing a comprehensive platform for investigating glioblastoma tumor microenvironment dynamics. The applications of this combined approach in studying glioblastoma dynamics are critically scrutinized, emphasizing its potential to bridge the gap between simplistic models and the intricate in vivo conditions. Furthermore, the article discusses the implications of biosensor-enhanced OOC models in elucidating the dynamic features of the tumor microenvironment, encompassing cell migration, proliferation, and interactions. By furnishing real-time insights, these models significantly contribute to unraveling the complex biology of glioblastoma, thereby influencing the development of more accurate diagnostic and therapeutic strategies.

Single-cell analysis of human prepuce reveals dynamic changes in gene regulation and cellular communications.

BACKGROUND: The cellular and molecular dynamics of human prepuce are crucial for understanding its biological and physiological functions, as well as the prevention of related genital diseases. However, the cellular compositions and heterogeneity of human prepuce at single-cell resolution are still largely unknown. Here we systematically dissected the prepuce of children and adults based on the single-cell RNA-seq data of 90,770 qualified cells. RESULTS: We identified 15 prepuce cell subtypes, including fibroblast, smooth muscle cells, T/natural killer cells, macrophages, vascular endothelial cells, and dendritic cells. The proportions of these cell types varied among different individuals as well as between children and adults. Moreover, we detected cell-type-specific gene regulatory networks (GRNs), which could contribute to the unique functions of related cell types. The GRNs were also highly dynamic between the prepuce cells of children and adults. Our cell-cell communication network analysis among different cell types revealed a set of child-specific (e.g., CD96, EPO, IFN-1, and WNT signaling pathways) and adult-specific (e.g., BMP10, NEGR, ncWNT, and NPR1 signaling pathways) signaling pathways. The variations of GRNs and cellular communications could be closely associated with prepuce development in children and prepuce maintenance in adults. CONCLUSIONS: Collectively, we systematically analyzed the cellular variations and molecular changes of the human prepuce at single-cell resolution. Our results gained insights into the heterogeneity of prepuce cells and shed light on the underlying molecular mechanisms of prepuce development and maintenance.

Elucidation of the interactome of the sucrose transporter StSUT4: sucrose transport is connected to ethylene and calcium signalling.

Sucrose transporters of the SUT4 clade show dual targeting to both the plasma membrane as well as to the vacuole. Previous investigations revealed a role for the potato sucrose transporter StSUT4 in flowering, tuberization, shade avoidance response, and ethylene production. Down-regulation of StSUT4 expression leads to early flowering, tuberization under long days, far-red light insensitivity, and reduced diurnal ethylene production. Sucrose export from leaves was increased and a phase-shift of soluble sugar accumulation in source leaves was observed, arguing for StSUT4 to be involved in the entrainment of the circadian clock. Here, we show that StSUT4, whose transcripts are highly unstable and tightly controlled at the post-transcriptional level, connects components of the ethylene and calcium signalling pathway. Elucidation of the StSUT4 interactome using the split ubiquitin system helped to prove direct physical interaction between the sucrose transporter and the ethylene receptor ETR2, as well as with the calcium binding potato calmodulin-1 (PCM1) protein, and a calcium-load activated calcium channel. The impact of calcium ions on transport activity and dual targeting of the transporter was investigated in detail. For this purpose, a reliable esculin-based transport assay was established for SUT4-like transporters. Site-directed mutagenesis helped to identify a diacidic motif within the seventh transmembrane spanning domain that is essential for sucrose transport activity and targeting, but not required for calcium-dependent inhibition. A link between sucrose, calcium and ethylene signalling has been previously postulated with respect to pollen tube growth, shade avoidance response, or entrainment of the circadian clock. Here, we provide experimental evidence for the direct interconnection of these signalling pathways at the molecular level by direct physical interaction of the main players.

Quantification of native mRNA dynamics in living neurons using fluorescence correlation spectroscopy and reduction-triggered fluorescent probes.

RNA localization in subcellular compartments is essential for spatial and temporal regulation of protein expression in neurons. Several techniques have been developed to visualize mRNAs inside cells, but the study of the behavior of endogenous and nonengineered mRNAs in living neurons has just started. In this study, we combined reduction-triggered fluorescent (RETF) probes and fluorescence correlation spectroscopy (FCS) to investigate the diffusion properties of activity-regulated cytoskeleton-associated protein (Arc) and inositol 1,4,5-trisphosphate receptor type 1 (Ip3r1) mRNAs. This approach enabled us to discriminate between RNA-bound and unbound fluorescent probes and to quantify mRNA diffusion parameters and concentrations in living rat primary hippocampal neurons. Specifically, we detected the induction of Arc mRNA production after neuronal activation in real time. Results from computer simulations with mRNA diffusion coefficients obtained in these analyses supported the idea that free diffusion is incapable of transporting mRNA of sizes close to those of Arc or Ip3r1 to distal dendrites. In conclusion, the combined RETF-FCS approach reported here enables analyses of the dynamics of endogenous, unmodified mRNAs in living neurons, affording a glimpse into the intracellular dynamics of RNA in live cells.

Troy+ brain stem cells cycle through quiescence and regulate their number by sensing niche occupancy.

The adult mouse subependymal zone provides a niche for mammalian neural stem cells (NSCs). However, the molecular signature, self-renewal potential, and fate behavior of NSCs remain poorly defined. Here we propose a model in which the fate of active NSCs is coupled to the total number of neighboring NSCs in a shared niche. Using knock-in reporter alleles and single-cell RNA sequencing, we show that the Wnt target Tnfrsf19/Troy identifies both active and quiescent NSCs. Quantitative analysis of genetic lineage tracing of individual NSCs under homeostasis or in response to injury reveals rapid expansion of stem-cell number before some return to quiescence. This behavior is best explained by stochastic fate decisions, where stem-cell number within a shared niche fluctuates over time. Fate mapping proliferating cells using a Ki67iresCreER allele confirms that active NSCs reversibly return to quiescence, achieving long-term self-renewal. Our findings suggest a niche-based mechanism for the regulation of NSC fate and number.

A Critical and Comparative Review of Fluorescent Tools for Live-Cell Imaging.

Fluorescent tools have revolutionized our ability to probe biological dynamics, particularly at the cellular level. Fluorescent sensors have been developed on several platforms, utilizing either small-molecule dyes or fluorescent proteins, to monitor proteins, RNA, DNA, small molecules, and even cellular properties, such as pH and membrane potential. We briefly summarize the impressive history of tool development for these various applications and then discuss the most recent noteworthy developments in more detail. Particular emphasis is placed on tools suitable for single-cell analysis and especially live-cell imaging applications. Finally, we discuss prominent areas of need in future fluorescent tool development-specifically, advancing our capability to analyze and integrate the plethora of high-content data generated by fluorescence imaging.

Cruciate ligament, patellar tendon, and patella formation involves differential cellular sources and dynamics as joint cavitation proceeds.

BACKGROUND: Cruciate ligament (CL) and patellar tendon (PT) are important elements of the knee joint, uniting femur, patella, and tibia into a single functional unit. So far, knowledge on the developmental mechanism of CL, PT, and patella falls far behind other skeletal tissues. RESULTS: Here, employing various lineage tracing strategies we investigate the cellular sources and dynamics that drive CL, PT, and patella formation during mouse embryonic development. We show that Gdf5 and Gli1 are generally expressed in the same cell population that only contributes to CL, but not PT or patella development. In addition, Col2 is expressed in two independent cell populations before and after joint cavitation, where the former contributes to the CL and the dorsal part of the PT and the latter contributes to the patella. Moreover, Prrx1 is always expressed in CL and PT progenitors, but not patella progenitors where it is switched off after joint cavitation. Finally, we reveal that patella development employs different cellular dynamics before and after joint cavitation. CONCLUSIONS: Our findings delineate the expression changes of several skeletogenesis-related genes before and after joint cavitation, and provide an indication on the cellular dynamics underlying ligament, tendon, and sesamoid bone formation during embryogenesis.

Molecular crosstalk: Notch can manipulate Hes1 and miR-9 behavior.

We propose a Hes1-Notch-miR-9 regulatory network and studied the regulating mechanism of miR-9 and Hes1 dynamics driven by Notch. Change in Notch concentration, which serves as a stress signal, can trigger the dynamics of Hes1 and miR-9 at five different states, namely, sTable (2), sustain (1) and mixed (2) states those may correspond to different cellular states. Further, this Notch stress signal introduce time reversal oscillation, which behaves as backward wave, after a certain threshold value of the stress signal and defends the system from moving to apoptosis. We also observe heterogeneous patterns of Hes1, miR-9 and other molecular species in various two dimensional parameter spaces and found that the variability in the patterns is triggered by Hill coefficient and Hes1 stress signal. The phase or bifurcation diagram in time period of oscillation (TN) driven by Notch signal provides all five states, predicts minimum threshold value TNc beyond which tendency to build up backward wave starts and TNc serves as bifurcation point of the system.

Regulation and plasticity of intestinal stem cells during homeostasis and regeneration.

The intestinal epithelium is the fastest renewing tissue in mammals and has a large flexibility to adapt to different types of damage. Lgr5+ crypt base columnar (CBC) cells act as stem cells during homeostasis and are essential during regeneration. Upon perturbation, the activity of CBCs is dynamically regulated to maintain homeostasis and multiple dedicated progenitor cell populations can reverse to the stem cell state upon damage, adding another layer of compensatory mechanisms to facilitate regeneration. Here, we review our current understanding of how intestinal stem and progenitor cells contribute to homeostasis and regeneration, and the different signaling pathways that regulate their behavior. Nutritional state and inflammation have been recently identified as upstream regulators of stem cell activity in the mammalian intestine, and we explore how these systemic signals can influence homeostasis and regeneration.

In vivo live imaging of bone cells.

There are as many as 200 cell types in the body, and highly sophisticated and varied life phenomena are carried out by cell migration to appropriate places at appropriate times following the appropriate interactions. Recent advances in optical imaging technology using multi-photon excitation microscopy have enabled visualization inside intact bone tissues in living animals without thin sectioning. Using such advanced techniques, the dynamic behaviors of living bone cells on intact bone tissue structures can be elucidated. Here, we focus on recent findings using intravital multi-photon imaging of dynamic biological systems, e.g., bone homeostasis. This novel approach has proven beneficial for understanding the mechanisms underlying the spatiotemporal nature of bone remodeling systems and for evaluating the specific modes of actions of novel drugs currently in development, which will contribute to a new chapter in bone and mineral research.

Dynamic behaviors of the non-neural ectoderm during mammalian cranial neural tube closure.

The embryonic brain and spinal cord initially form through the process of neural tube closure (NTC). NTC is thought to be highly similar between rodents and humans, and studies of mouse genetic mutants have greatly increased our understanding of the molecular basis of NTC with relevance for human neural tube defects. In addition, studies using amphibian and chick embryos have shed light into the cellular and tissue dynamics underlying NTC. However, the dynamics of mammalian NTC has been difficult to study due to in utero development until recently when advances in mouse embryo ex vivo culture techniques along with confocal microscopy have allowed for imaging of mouse NTC in real time. Here, we have performed live imaging of mouse embryos with a particular focus on the non-neural ectoderm (NNE). Previous studies in multiple model systems have found that the NNE is important for proper NTC, but little is known about the behavior of these cells during mammalian NTC. Here we utilized a NNE-specific genetic labeling system to assess NNE dynamics during murine NTC and identified different NNE cell behaviors as the cranial region undergoes NTC. These results bring valuable new insight into regional differences in cellular behavior during NTC that may be driven by different molecular regulators and which may underlie the various positional disruptions of NTC observed in humans with neural tube defects.

SIRT6 knockout cells resist apoptosis initiation but not progression: a computational method to evaluate the progression of apoptosis.

Apoptosis is essential for numerous processes, such as development, resistance to infections, and suppression of tumorigenesis. Here, we investigate the influence of the nutrient sensing and longevity-assuring enzyme SIRT6 on the dynamics of apoptosis triggered by serum starvation. Specifically, we characterize the progression of apoptosis in wild type and SIRT6 deficient mouse embryonic fibroblasts using time-lapse flow cytometry and computational modelling based on rate-equations and cell distribution analysis. We find that SIRT6 deficient cells resist apoptosis by delaying its initiation. Interestingly, once apoptosis is initiated, the rate of its progression is higher in SIRT6 null cells compared to identically cultured wild type cells. However, SIRT6 null cells succumb to apoptosis more slowly, not only in response to nutrient deprivation but also in response to other stresses. Our data suggest that SIRT6 plays a role in several distinct steps of apoptosis. Overall, we demonstrate the utility of our computational model to describe stages of apoptosis progression and the integrity of the cellular membrane. Such measurements will be useful in a broad range of biological applications.

Neuropeptide substance P and the immune response.

Substance P is a peptide mainly secreted by neurons and is involved in many biological processes, including nociception and inflammation. Animal models have provided insights into the biology of this peptide and offered compelling evidence for the importance of substance P in cell-to-cell communication by either paracrine or endocrine signaling. Substance P mediates interactions between neurons and immune cells, with nerve-derived substance P modulating immune cell proliferation rates and cytokine production. Intriguingly, some immune cells have also been found to secrete substance P, which hints at an integral role of substance P in the immune response. These communications play important functional roles in immunity including mobilization, proliferation and modulation of the activity of immune cells. This review summarizes current knowledge of substance P and its receptors, as well as its physiological and pathological roles. We focus on recent developments in the immunobiology of substance P and discuss the clinical implications of its ability to modulate the immune response.

Engineered temperature compensation in a synthetic genetic clock.

Synthetic biology promises to revolutionize biotechnology by providing the means to reengineer and reprogram cellular regulatory mechanisms. However, synthetic gene circuits are often unreliable, as changes to environmental conditions can fundamentally alter a circuit's behavior. One way to improve robustness is to use intrinsic properties of transcription factors within the circuit to buffer against intra- and extracellular variability. Here, we describe the design and construction of a synthetic gene oscillator in Escherichia coli that maintains a constant period over a range of temperatures. We started with a previously described synthetic dual-feedback oscillator with a temperature-dependent period. Computational modeling predicted and subsequent experiments confirmed that a single amino acid mutation to the core transcriptional repressor of the circuit results in temperature compensation. Specifically, we used a temperature-sensitive lactose repressor mutant that loses the ability to repress its target promoter at high temperatures. In the oscillator, this thermoinduction of the repressor leads to an increase in period at high temperatures that compensates for the decrease in period due to Arrhenius scaling of the reaction rates. The result is a transcriptional oscillator with a nearly constant period of 48 min for temperatures ranging from 30 °C to 41 °C. In contrast, in the absence of the mutation the period of the oscillator drops from 60 to 30 min over the same temperature range. This work demonstrates that synthetic gene circuits can be engineered to be robust to extracellular conditions through protein-level modifications.

Intravital imaging technology reveals immune system dynamics in vivo.

Fluorescent 'intravital' imaging is a new research technique by which the interior of living tissues and organs (in living bodies, if possible) can be observed, revealing the kinetics of cell and molecular processes in real time. Recent technological innovations in optical equipment and fluorescence imaging techniques have enabled a variety of cellular phenomena in different tissues and organs to be characterized under completely native conditions. This shift from static to dynamic biology constitutes the beginning of a new era in biomedical sciences.