Clearing techniques for deeper imaging of plants and plant-microbe interactions.

Plant cells are uniquely characterized by exhibiting cell walls, pigments, and phenolic compounds, which can impede microscopic observations by absorbing and scattering light. The concept of clearing was first proposed in the late nineteenth century to address this issue, aiming to render plant specimens transparent using chloral hydrate. Clearing techniques involve chemical procedures that render biological specimens transparent, enabling deep imaging without physical sectioning. Drawing inspiration from clearing techniques for animal specimens, various protocols have been adapted for plant research. These procedures include (i) hydrophobic methods (e.g., Visikol™), (ii) hydrophilic methods (ScaleP and ClearSee), and (iii) hydrogel-based methods (PEA-CLARITY). Initially, clearing techniques for plants were mainly utilized for deep imaging of seeds and leaves of herbaceous plants such as Arabidopsis thaliana and rice. Utilizing cell wall-specific fluorescent dyes for plants and fungi, researchers have documented the post-penetration behavior of plant pathogenic fungi within hosts. State-of-the-art plant clearing techniques, coupled with microbe-specific labeling and high-throughput imaging methods, offer the potential to advance the in planta characterization of plant microbiomes.

Erratum: Assessment Methods for Problematic Eating Behaviors in Children and Adolescents With Autism Spectrum Disorder.

[This corrects the article on p. 57 in vol. 35, PMID: 38204745.].

Assessment Methods for Problematic Eating Behaviors in Children and Adolescents With Autism Spectrum Disorder.

Autism spectrum disorder (ASD) can be associated with eating problems. However, currently, there is a lack of established guidelines for assessing and addressing eating behaviors in individuals with ASD. This gap in research exists due to the challenges associated with using traditional assessment methods, which may lead to discrepancies in responses and unintentional potential biases from caregivers. In this review, we provided a comprehensive overview of various eating behaviors commonly observed in individuals with ASD. These behaviors include 1) food neophobia, 2) selective eating, 3) binge eating, 4) food avoidance, 5) chewing and swallowing problems, 6) pica, 7) rumination, 8) rituals, and 9) problematic behaviors. Furthermore, we provide a perspective of utilizing digital tools: 1) augmentative and alternative communication; 2) ecological momentary assessment; and 3) video analysis, behavioral analysis, and facial expression analysis. This review explores existing assessment methods and suggests novel assessment aiding together.

Astrocytic FoxO1 in the hypothalamus regulates metabolic homeostasis by coordinating neuropeptide Y neuron activity.

The forkhead box transcription factor O1 (FoxO1) is expressed ubiquitously throughout the central nervous system, including in astrocytes, the most prevalent glial cell type in the brain. While the role of FoxO1 in hypothalamic neurons in controlling food intake and energy balance is well-established, the contribution of astrocytic FoxO1 in regulating energy homeostasis has not yet been determined. In the current study, we demonstrate the essential role of hypothalamic astrocytic FoxO1 in maintaining normal neuronal activity in the hypothalamus and whole-body glucose metabolism. Inhibition of FoxO1 function in hypothalamic astrocytes shifts the cellular metabolism from glycolysis to oxidative phosphorylation, enhancing astrocyte ATP production and release meanwhile decreasing astrocytic export of lactate. As a result, specific deletion of astrocytic FoxO1, particularly in the hypothalamus, causes a hyperactivation of hypothalamic neuropeptide Y neurons, which leads to an increase in acute feeding and impaired glucose regulation and ultimately results in diet-induced obesity and systemic glucose dyshomeostasis.

Morphological remodeling of Coxiella burnetii during its biphasic developmental cycle revealed by cryo-electron tomography.

Coxiella burnetii is an obligate zoonotic bacterium that targets macrophages causing a disease called Q fever. It has a biphasic developmental life cycle where the extracellular and metabolically inactive small cell variant (SCV) transforms inside the host into the vegetative large cell variant (LCV). However, details about the morphological and structural changes of this transition are still lacking. Here, we used cryo-electron tomography to image both SCV and LCV variants grown either under axenic conditions or purified directly from host cells. We show that SCVs are characterized by equidistant stacks of inner membrane that presumably facilitate the transition to LCV, a transition coupled with the expression of the Dot/Icm type IVB secretion system (T4BSS). A class of T4BSS particles were associated with extracellular densities possibly involved in host infection. Also, SCVs contained spherical multilayered membrane structures of different sizes and locations suggesting no connection to sporulation as once assumed.

Mitochondria-derived peptide SHLP2 regulates energy homeostasis through the activation of hypothalamic neurons.

Small humanin-like peptide 2 (SHLP2) is a mitochondrial-derived peptide implicated in several biological processes such as aging and oxidative stress. However, its functional role in the regulation of energy homeostasis remains unclear, and its corresponding receptor is not identified. Hereby, we demonstrate that both systemic and intracerebroventricular (ICV) administrations of SHLP2 protected the male mice from high-fat diet (HFD)-induced obesity and improved insulin sensitivity. In addition, the activation of pro-opiomelanocortin (POMC) neurons by SHLP2 in the arcuate nucleus of the hypothalamus (ARC) is involved in the suppression of food intake and the promotion of thermogenesis. Through high-throughput structural complementation screening, we discovered that SHLP2 binds to and activates chemokine receptor 7 (CXCR7). Taken together, our study not only reveals the therapeutic potential of SHLP2 in metabolic disorders but also provides important mechanistic insights into how it exerts its effects on energy homeostasis.

Blockade of Activin Receptor IIB Protects Arthritis Pathogenesis by Non-Amplification of Activin A-ACVR2B-NOX4 Axis Pathway.

Although activin receptor IIB (ACVR2B) is emerging as a novel pathogenic receptor, its ligand and assembled components (or assembly) are totally unknown in the context of osteoarthritis (OA) pathogenesis. The present results suggest that upregulation of ACVR2B and its assembly could affect osteoarthritic cartilage destruction. It is shown that the ACVR2B ligand, activin A, regulates catabolic factor expression through ACVR2B in OA development. Activin A Tg mice (Col2a1-Inhba) exhibit enhanced cartilage destruction, whereas heterozygous activin A KO mice (Inhba+/- ) show protection from cartilage destruction. In silico analysis suggests that the Activin A-ACVR2B axis is involved in Nox4-dependent ROS production. Activin A Tg:Nox4 KO (Col2a1-Inhba:Nox4-/- ) mice show inhibition of experimental OA pathogenesis. NOX4 directly binds to the C-terminal binding site on ACVR2B-ACVR1B and amplifies the pathogenic signal for cartilage destruction through SMAD2/3 signaling. Together, the findings reveal that the ACVR2B assembly, which comprises Activin A, ACVR2B, ACVR1B, Nox4, and AP-1-induced HIF-2α, accelerates OA development. Furthermore, it is shown that shRNA-mediated ACVR2B knockdown or trapping ligands of ACVR2B abrogate OA development by competitively disrupting the ACVR2B-Activin A interaction. These results suggest that the ACVR2B assembly is required to amplify osteoarthritic cartilage destruction and could be a potential therapeutic target in efforts to treat OA.

Light and scanning electron microscopy of aecia and aeciospores of Cronartium ribicola on Pinus koraiensis branch tissues.

Morphological characteristics of aecia and aeciospores of Cronartium ribicola on Pinus koraiensis branch tissues were investigated using light and field emission scanning electron microscopy (FESEM). Mature P. koraiensis trees in Jeongseon, Korea, showed yellowish aecia on stems and branches. Aecia and surrounding tissues were excised from the lesions and vapor-fixed for FESEM imaging, which revealed morphology including intact blister-shaped, flattened, and burst forms. Light microscopy revealed yellowish aeciospores having surface projections. Aeciospores were mostly ovoid and measured approximately 20 µm long. The FESEM showed irregularly shaped cracks on the aecia that had erupted through the bark of P. koraiensis. Some aeciospores had germinated, producing two germ tubes from a spore in a burst aecium. Aeciospores had both smooth and verrucose regions on the surface, and some had concave or convex regions. Aeciospore layers and underlying fungal matrices including aecial columns were obvious in the cross-sections of aecia. Approximately 1 µm-high wart-like surface projections could be resolved and comprised less than 10 angular platelets stacked in vertical rows. Remains of the primary spore wall were present between surface projections. These results provide insights into the morphology of the heteroecious rust fungus with the help of vapor fixation and high-resolution surface imaging.

Protective effect of TPP-Niacin on microgravity-induced oxidative stress and mitochondrial dysfunction of retinal epithelial cells.

Adverse effects of spaceflight on the human body are attritubuted to microgravity and space radiation. One of the most sensitive organs affected by them is the eye, particularly the retina. The conditions that astronauts suffer, such as visual acuity, is collectively called a spaceflight-associated neuro-ocular syndrome (SANS); however, the underlying molecular mechanism of the microgravity-induced ocular pathogenesis is not clearly understood. The current study explored how microgravity affects the retina function in ARPE19 cells in vitro under time-averaged simulated microgravity (µG) generated by clinostat. We found multicellular spheroid (MCS) formation and a significantly decreased cell migration potency under µG conditions compared to 1G in ARPE19 cells. We also observed that µG increases intracellular reactive oxygen species (ROS) and causes mitochondrial dysfunction in ARPE19 cells. Subsequently, we showed that µG activates autophagic pathways and ciliogenesis. Furthermore, we demonstrated that mitophagy activation is triggered via the mTOR-ULK1-BNIP3 signaling axis. Finally, we validated the effectiveness of TPP-Niacin in mitigating µG-induced oxidative stress and mitochondrial dysfunction in vitro, which provides the first experimental evidence for TPP-Niacin as a potential therapeutic agent to ameliorate the cellular phenotypes caused by µG in ARPE19 cells. Further investigations are, however, required to determine its physiological functions and biological efficacies in primary human retinal cells, in vivo models, and target identification.

Appressed, directed and silicified trichomes of the kudzu climbing vine.

Kudzu (Pueraria montana var. lobata) is becoming one of the most prevalent climbing vines in urban forests. Here, surface characteristics of kudzu leaves and stems are investigated using field-emission scanning electron microscopy and X-ray microanalysis. The leaf and stem surfaces were characterized by different types of trichomes. No adhesive pads, modified hooks or tendrils were found on kudzu vines. Trichomes on the leaves and stems of kudzu could be categorized into (i) complex bulbous glandular trichomes and (ii) needle-shaped nonglandular trichomes (NSNGTs). Two morphotypes of the nonglandular trichomes were present on the stems: (i) long hairy nonglandular trichomes and (ii) short NSNGTs. The leaf trichomes were appressed and oriented in an uphill direction, whereas the stem trichomes were appressed and oriented in a downhill direction. This opposite trichome orientation appears to facilitate climbing and prohibits slipping of kudzu vines through differential friction between the plants and the kudzu vines. X-ray microanalysis revealed a distinct accumulation of silicon in the leaf and stem trichomes. These results suggest that appressed, directed and silicified trichomes may contribute to the twining behavior of kudzu vines.

Primary cilia regulate adaptive responses to fasting.

OBJECTIVE: Neuronal primary cilia are known to be a required organelle for energy balance and leptin action. However, whether primary cilia directly mediate adaptive responses during starvation is yet unknown. Therefore, we investigated the counterregulatory roles of primary cilia, and their related leptin action in energy-depleted condition. METHOD: We generated leptin receptor (LepR) neuron-specific primary cilia knockout (Ift88 KOLepR) mice. Leptin-mediated electrophysiological properties of the neurons in fasting condition were assessed using patch-clamp technique. Adaptive responses and neuroendocrine reflexes were measured by monitoring counterregulatory hormones. RESULTS: In fasting state, the leptin-induced neuronal excitability and leptin homeostasis were impaired in Ift88 KOLepR. In addition, the Ift88 KOLepR exhibited aberrant fasting responses including lesser body weight loss, decreased energy expenditure, and lower heat generation compared to wild-type littermates. Furthermore, the primary cilia in LepR neurons are necessary for counterregulatory responses and leptin-mediated neuroendocrine adaptation to starvation. CONCLUSION: Our results demonstrated that the neuronal primary cilia are crucial neuronal components mediating the adaptive counterregulatory responses to starvation.

Biological applications of the NanoSuit for electron imaging and X-microanalysis of insulating specimens.

Field emission scanning electron microscopy (FESEM) is an essential tool for observing surface details of specimens in a high vacuum. A series of specimen procedures precludes the observations of living organisms, resulting in artifacts. To overcome these problems, Takahiko Hariyama and his colleagues proposed the concept of the "nanosuit" later referred to as "NanoSuit", describing a thin polymer layer placed on organisms to protect them in a high vacuum in 2013. The NanoSuit is formed rapidly by (i) electron beam irradiation, (ii) plasma irradiation, (iii) Tween 20 solution immersion, and (iv) surface shield enhancer (SSE) solution immersion. Without chemical fixation and metal coating, the NanoSuit-formed specimens allowed structural preservation and accurate element detection of insulating, wet specimens at high spatial resolution. NanoSuit-formed larvae were able to resume normal growth following FESEM observation. The method has been employed to observe unfixed and uncoated bacteria, multicellular organisms, and paraffin sections. These results suggest that the NanoSuit can be applied to prolong life in vacuo and overcome the limit of dead imaging of electron microscopy.

X-ray computed tomography, electron microscopy, and energy-dispersive X-ray spectroscopy of severed Zelkova serrata roots (Japanese elm tree).

X-ray computed tomography (XCT), field emission scanning electron microscopy (FESEM), and energy-dispersive X-ray spectroscopy (EDS) were evaluated for imaging and element identification of woody plant roots. Lateral roots of Japanese zelkova (Zelkova serrata) were severed in spring and maintained in soil for six months. The lateral roots were observed using XCT without maceration and sectioning. The general wood characteristics were discernible to reveal the bark and xylem structures in contrast-inverted tomograms. Virtual sections showed a newly formed ring of woundwood encircling the severed lateral roots. FESEM exhibited secondary xylem structures in which tyloses, fungal hyphae, and aggregates were present. While silicon was dispersed in and around the fungal hyphae, calcium was localized as distinct aggregates using EDS. These results suggest that the combined use of XCT, FESEM, and EDS has merit into the morphological assessment of tree health care, providing virtual sections, high-resolution images, and element composition from an entire woodblock.

Fine-tuning of epithelial taste bud organoid to promote functional recapitulation of taste reactivity.

Taste stem/progenitor cells from posterior mouse tongues have been used to generate taste bud organoids. However, the inaccessible location of taste receptor cells is observed in conventional organoids. In this study, we established a suspension-culture method to fine-tune taste bud organoids by apicobasal polarity alteration to form the accessible localization of taste receptor cells. Compared to conventional Matrigel-embedded organoids, suspension-cultured organoids showed comparable differentiation and renewal rates to those of taste buds in vivo and exhibited functional taste receptor cells and cycling progenitor cells. Accessible taste receptor cells enabled the direct application of calcium imaging to evaluate the taste response. Moreover, suspension-cultured organoids can be genetically altered. Suspension-cultured taste bud organoids harmoniously integrated with the recipient lingual epithelium, maintaining the taste receptor cells and gustatory innervation capacity. We propose that suspension-cultured organoids may provide an efficient model for taste research, including taste bud development, regeneration, and transplantation.

Hypothalamic control of energy expenditure and thermogenesis.

Energy expenditure and energy intake need to be balanced to maintain proper energy homeostasis. Energy homeostasis is tightly regulated by the central nervous system, and the hypothalamus is the primary center for the regulation of energy balance. The hypothalamus exerts its effect through both humoral and neuronal mechanisms, and each hypothalamic area has a distinct role in the regulation of energy expenditure. Recent studies have advanced the understanding of the molecular regulation of energy expenditure and thermogenesis in the hypothalamus with targeted manipulation techniques of the mouse genome and neuronal function. In this review, we elucidate recent progress in understanding the mechanism of how the hypothalamus affects basal metabolism, modulates physical activity, and adapts to environmental temperature and food intake changes.

Some living eukaryotes during and after scanning electron microscopy.

Electron microscopy (EM) is an essential imaging method in biological sciences. Since biological specimens are exposed to radiation and vacuum conditions during EM observations, they die due to chemical bond breakage and desiccation. However, some organisms belonging to the taxa of bacteria, fungi, plants, and animals (including beetles, ticks, and tardigrades) have been reported to survive hostile scanning EM (SEM) conditions since the onset of EM. The surviving organisms were observed (i) without chemical fixation, (ii) after mounting to a precooled cold stage, (iii) using cryo-SEM, or (iv) after coating with a thin polymer layer, respectively. Combined use of these techniques may provide a better condition for preservation and live imaging of multicellular organisms for a long time beyond live-cell EM.

Teliospore mucilage of Puccinia miscanthi revealed through the axial imaging of secondary electrons.

Puccinia miscanthi teliospores were observed on the leaf surface of Miscanthus sinensis using a field emission scanning electron microscope. Details of teliospore mucilage could be visualized through the axial imaging of secondary electrons for a better understanding of pathogen behavior in rust diseases.

Evaluation of electroporated area using 2,3,5-triphenyltetrazolium chloride in a potato model.

Irreversible electroporation (IRE) is a tissue ablation method, uses short high electric pulses and results in cell death in target tissue by irreversibly permeabilizing the cell membrane. Potato is commonly used as a tissue model for electroporation experiments. The blackened area that forms 12 h after electric pulsing is regarded as an IRE-ablated area caused by melanin accumulation. Here, the 2,3,5-triphenyltetrazolium chloride (TTC) was used as a dye to assess the IRE-ablated area 3 h after potato model ablation. Comparison between the blackened area and TTC-unstained white area in various voltage conditions showed that TTC staining well delineated the IRE-ablated area. Moreover, whether the ablated area was consistent over time and at different staining times was investigated. In addition, the presumed reversible electroporation (RE) area was formed surrounding the IRE-ablated area. Overall, TTC staining can provide a more rapid and accurate electroporated area evaluation.

Hypothalamic primary cilium: A hub for metabolic homeostasis.

Obesity is a global health problem that is associated with adverse consequences such as the development of metabolic disorders, including cardiovascular disease, neurodegenerative disorders, and type 2 diabetes. A major cause of obesity is metabolic imbalance, which results from insufficient physical activity and excess energy intake. Understanding the pathogenesis of obesity, as well as other metabolic disorders, is important in the development of methods for prevention and therapy. The coordination of energy balance takes place in the hypothalamus, a major brain region that maintains body homeostasis. The primary cilium is an organelle that has recently received attention because of its role in controlling energy balance in the hypothalamus. Defects in proteins required for ciliary function and formation, both in humans and in mice, have been shown to cause various metabolic disorders. In this review, we provide an overview of the critical functions of primary cilia, particularly in hypothalamic areas, and briefly summarize the studies on the primary roles of cilia in specific neurons relating to metabolic homeostasis.