Fluorescent Indicator Research Articles

STEM-16. EXPLORING THE ROLE OF HYPOXIA AND MACROPHAGES ON GLIOBLASTOMA QUIESCENCE AND STEMNESS AS MEDIATORS OF RADIORESISTANCE

Abstract Our ultimate goal is to identify, characterize and therapeutically target the glioma cell population that is responsible for treatment resistance and recurrence following chemoradiation. Two cell types that may have overlapping phenotypes and are known to influence radioresistance are 1) quiescent cells and 2) glioblastoma (GBM) stem cells (GSCs). The extent of overlap between these populations is unknown as are the influence of hypoxia and immune cells on their radioresistant properties. We have shown that hypoxia and co-culturing with THP-1 macrophages dramatically increases the percentage of GSCs. Using the Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) system expressed in patient-derived gliomas, we used flow cytometry to distinguish cells based on their cell cycle status (G1/G0 and G2/M) and classified them as quiescent (high p27, low Ki67) or GSC (high CD133/CD44 co-expression). We found that the fraction of cells in G0 expressed high p27 and low Ki67, indicating cell cycle suppression and low proliferation, respectively. G0 glioma cells also contained a high percentage of GSCs, but GSCs were also distributed in G1 and G2/M. Following exposure to hypoxia, flow cytometry of FUCCI GBM cells showed an increase of cells in G0. After 3 Gy radiation under normoxic conditions, GBM cells demonstrated a shift to increased levels of G2/M cells compared to non-irradiated cells. In hypoxia, however, GBM cells sustained their population of cells in G0 and did not exhibit such cell cycle shifts. Our results indicate that both hypoxia and macrophages cause GSC enrichment and that hypoxia increases the proportion of quiescent cells in G0. Additional study is necessary to determine the extent of overlap between the GSCs and quiescent cells and to identify the intrinsic cell properties that drive tumor recurrence.

Multifunctional Neural Probes Enable Bidirectional Electrical, Optical, and Chemical Recording and Stimulation In Vivo.

Recording and modulation of neuronal activity enables the study of brain function in health and disease. While translational neuroscience relies on electrical recording and modulation techniques, mechanistic studies in rodent models leverage genetic precision of optical methods, such as optogenetics and fluorescent indicator imaging. In addition to electrical signal transduction, neurons produce and receive diverse chemical signals which motivate tools to probe and modulate neurochemistry. Although the past decade has delivered a wealth of technologies for electrophysiology, optogenetics, chemical sensing, and optical recording, combining these modalities within a single platform remains challenging. This work leverages materials selection and convergence fiber drawing to permit neural recording, electrical stimulation, optogenetics, fiber photometry, drug and gene delivery, and voltammetric recording of neurotransmitters within individual fibers. Composed of polymers and non-magnetic carbon-based conductors, these fibers are compatible with magnetic resonance imaging, enabling concurrent stimulation and whole-brain monitoring. Their utility is demonstrated in studies of the mesolimbic reward pathway by interfacing with the ventral tegmental area and nucleus accumbens in mice and characterizing the neurophysiological effects of a stimulant drug. This study highlights the potential of these fibers to probe electrical, optical, and chemical signaling across multiple brain regions in both mechanistic and translational studies.

A ratiometric fluorescence platform based on bifunctional MOFs for sensitive detection of organophosphorus pesticides

Rapid and precise detection of organophosphorus pesticides (OPs) is of great importance for life health. Herein, a new sensing platform is developed through a dual signal amplification approach for sensitive quantification of OPs, with a bimetallic metal organic framework (UiO-66(Zr/Fe)-NH2) as peroxide-like nanozyme and fluorescent indicator. In the presence of thiocholine (TCh), the hydrolyzate of acetylthiocholine iodide catalyzed by acetylcholinesterase, the catalytic oxidization of o-phenylenediamine by UiO-66(Fe/Zr)-NH2 to strong fluorescent 2,3-diaminophenazine (λem 555nm) is significantly inhibited. OPs-triggered acetylcholinesterase inactivation enables the renaturation of UiO-66(Fe/Zr)-NH2. Interestingly, the introduction of 1,4-dioxane further boosts the amplification of fluorescence signals. By virtue of the recovered fluorescence at 555nm and MOF fluorescence (λem 446nm), the dual signal amplification-based ratiometric fluorescent platform is successfully employed for the selective recognition and sensitive detection of OPs. This work is expected to provide a facile and sensitive method for accurate OPs assay, and make a positive contribution to food safety.

UV light-triggered fluorescence corrosion sensing coatings for AA2024-T3 based on 8-hydroxyquinline loaded vanadium oxide nanorods

Fluorescent corrosion indicators potentially locate the underlying corrosion on metallic substrates based on sensing metal ions caused by corrosion reactions. Nanoparticles loaded with indicators are significant components of the fluorescence corrosion detection system. These fluorescent coatings face challenges (including fluorescence quenching, sluggish response rate, and expensive instrumentation). To tackle such issues, we have developed 8-hydroxyquinoline (8-HQ) loaded oxygen-deficient vanadium oxide (VOx) nanorods (∼220 nm in length and ∼50 nm in width) via solvothermal technique yielding enhanced fluorescence efficiency. 8-HQ/VOx/Ac2403 was utilized to develop a fluorescent corrosion sensing coating for AA2024-T3. The system is designed to detect corrosion within the first few days to weeks after exposure to corrosive conditions by making a complex by reacting with metal ions (Al3+), resulting in a reduction and bright blueish fluorescence effect in the exposure of UV light and can be seen with the naked eye. As a result, fluorescent corrosion-sensing coatings provide a practical substitute for typical coatings to satisfy rising industrial demands by providing corrosion sensing before significant structural damage occurs.

PET nano-woven luminescent fibers for efficient air filtration and fluorescence indication of particulate matter

Inhalable particulate matter poses significant health risks, and current air filtration materials often struggle with efficiently capturing particles smaller than 300 nm and providing real-time feedback on filtration performance. We present the synthesis and characterization of polyethylene terephthalate (PET) nano-woven luminescent fibers (PNWLFs), created through a positive and negative dual high-voltage electrospinning method. The PNWLFs feature a unique nano-woven structure composed of backbone fibers (200–1000 nm) and nanofibers (20–160 nm), which endow them with remarkable mechanical strength, maintaining a breaking force of 117.5 cN and structural integrity under a 138 mm water column. The fibers exhibit excellent breathability and waterproof performance, with a water vapor transmission rate of 4.69 kg/m2/day. More importantly, the PNWLFs demonstrate exceptional air filtration capabilities, achieving filtration efficiencies of 99.77 % for PM5.0, 99.76 % for PM2.5, 99.72 % for PM1.0, 99.53 % for PM0.5, and 99.40 % for PM0.3, with minimal pressure drop of 106 Pa. Additionally, these fibers possess a unique fluorescence-indicating function: the fluorescence intensity decreases in response to increasing particulate matter concentration and the number of filtration cycles, allowing for intuitive visual monitoring of filter performance and particulate levels. These attributes underscore the significant potential of PNWLFs synthesized by this work for advanced air filtration applications and effective air quality monitoring.

Asymptomatic bacteriuria screening for developing countries using a modified water quality test kit.

Between 2% and 15% of pregnant women unknowingly experience asymptomatic bacteriuria (ASB), defined as ≥105 CFU per milliliter of urine in the absence of symptoms. ASB increases the risk of adverse pregnancy outcomes including pyelonephritis, preterm labor, and low-birth weight infants. While pregnant women in the United States are routinely screened for ASB, those in developing countries with limited resources and funding lack an accurate mechanism for ASB screening. Aquagenx water quality test kits detect Escherichia coli, the most common causative agent of ASB, and total coliform bacteria in drinking water via colorimetric and fluorescent indicators. We found that the Aquagenx system is compatible with human urine and then proceeded to develop an ASB screening protocol using disposable inoculating loops. Our protocol diagnosed artificial ASB- samples (104 CFU/mL E. coli) with a false positive (FP) rate of 33% (n = 18) and ASB+ (105 CFU/mL E. coli) with a false negative (FN) rate of 5.6% (n = 18). Clinical sample testing with our protocol revealed a FP rate of 0% in ASB- samples (n = 28) and a FN rate of 0% in ASB+ samples caused by coliforms (n = 13). Aquagenx did not detect ASB in nine clinical samples with non-coliform etiological agents due to the limitations of the technology. However, with very high accuracy for detection of E. coli and other causative agents that collectively account for 90.1% of ASB cases, these kits could be used as a diagnostic ASB screening tool in developing countries in which there is currently no alternative to urine culture.IMPORTANCEAsymptomatic bacteriuria (ASB) affects 2%-15% of pregnant women and can result in adverse maternal and fetal outcomes if left undetected and untreated. In the United States and other developed nations, pregnant women are regularly screened for ASB via urine culture. However, in low-resource countries where bacterial culture is not available, dipstick testing is used. Although accurate in cases of symptomatic bacteriuria, dipstick detection is ineffective for detecting ASB. Here, we made use of an existing water quality field test for ASB urine screening, which would be readily deployable in low-resource settings. We optimized a dilution protocol for sampling patient urine within the detection limits of the Aquagenx kit technology. Overall, we were able to detect ASB samples with Gram-negative pathogens that collectively account for 90% of all ASB cases. Utilization of this repurposed technology for proactive medical screening may help prevent adverse pregnancy and birth outcomes due to ASB.

Recent Development of Chemigenetic Hybrid Voltage Indicators Enabled by Bioconjugation Chemistry.

Fluorescent voltage indicators enable the optical recording of electrophysiology across large cell populations with subcellular resolution; however, their application is often constrained by a limited photon budget. To address this limitation, advanced bioconjugation methods have been employed to site-specifically attach bright and photostable organic dyes to cell-specific protein scaffolds in live cells. The resulting chemigenetic hybrid voltage indicators enable sustained monitoring of voltage fluctuations with an exceptional signal-to-noise ratio, both in vitro and in vivo. This Viewpoint discusses recent advancements in the development of these indicators through bioconjugation chemistry.

SMAD3 silencing induces Epithelial-to-Mesenchymal Transition (EMT) towards cardiac pericyte progenitors with proangiogenic potential in hiPSC-derived epicardial cells

Abstract Background The epicardium is only a single mesothelial layer and "quiescent" in the adult heart. However, once the heart is injured, the epicardium reactivates and acts as a key for cardiac regeneration through epithelium-to-mesenchymal transition (EMT). While the canonical TGF-β-SMAD pathway plays a central role in regulating EMT, SMAD3 functions independent of this pathway in epicardium remain unknown. Purpose The purpose of this study is to investigate the effects of SMAD3 downregulation on EMT in epicardial cells. Methods First, we differentiated human iPSC-derived epicardial cells (EPI cells) by mimicking the process of cardiac mesoderm development. Next, siRNA targeting SMAD3 was transfected into EPI cells to silence SMAD3. We performed qRT-PCR, western blotting, immunocytochemistry, flow cytometry, transcriptional analysis, endothelial tube formation assay, and paracrine experiments using FUCCI (fluorescent ubiquitination-based cell cycle indicator) system to evaluate the phenotype of EPI cells with silenced SMAD3. Results We found that SMAD3 silencing in EPI cells resulted in loss of epicardial identity and upregulation of multiple EMT markers. Additionally, NG2 and ENG (CD105), both cardiac pericyte markers, were highly expressed in SMAD3-silenced EPI cells. RNA-sequence also revealed that SMAD3 silencing in EPI cells induced EMT facilitated towards cardiac pericyte progenitors with proangiogenic potential. In endothelial tube formation assay, SMAD3-silenced EPI cells demonstrated enhanced tube formation compared to the control (1.48-fold increase in the number of junctions, n=3; p<0.0001), which indicated their angiogenic potential. At last, we revealed the paracrine effect of SMAD3-silenced EPI cells to enhance the proliferative reactivation in iPSC-derived cardiomyocytes by showing upregulation in CDK6/CCND1 axis and FUCCI-green. Conclusion Our findings demonstrate a new role of SMAD3 biology in the human epicardium, specifically in the generation of cardiac pericyte progenitor cells that contribute to better responses in angiogenesis of the heart microvasculature enabling the possibility to continue exploiting epicardial biology for effective cardiac regeneration.

Utilizing fluorescence indicators to apportion organic sources in estuarine/coastal sediments: A comparison with a stable isotopic model

Coastal sediments accumulate organic matter (OM) from diverse sources, including local anthropogenic pollution. Effective source tracking of sediment OM is crucial for pollution source management. This study compares fluorescence proxies and stable isotopic ratios as tracers for sediment OM in Gangu Port, Korea. An optimized extraction method using distilled water for 0.5 h yielded distinct fluorescence signatures. The humification index (HIX) and protein-like component (C3%) showed ideal mixing behavior with two end-members (fishery market sediment and algae). A Bayesian end-member mixing analysis model revealed that agricultural soil is the prevalent contributor to sediments, aligning with land use patterns. The fluorescence-based model showed higher sensitivity to anthropogenic influences compared to traditional stable isotope ratios. The results strongly agreed with isotope ratio-based predictions, exhibiting a positive correlation (p < 0.05) at 8 out of 14 sites. This study highlights the potential of fluorescence-based tracking to complement or replace conventional stable isotope methods.

Effect of Bacillus subtilis – based microbiological preparations on the numerical composition of cultured forms of endophytic bacteria, content of mineral nutrition elements, photosynthetic pigments, as well as on chlorophyll fluorescence indices in leaves of columnar apple trees

The results of agrochemical field experiments conducted in an orchard of columnar apple tree (Malus domestica Borkh.) in the Moscow Oblast during the period of 2021–2022 are presented. The research objects were the ‘Valuta’, ‘Triumph’, and ‘President’ varieties. The effect of foliar treatments of plants with Bacillus subtilis (strain V167) and Bacillus subtilis (strain V417) strains, as well as Bacillus subtilis – based microbiological preparations “Extrasol” (strain Ch13) and “Phytosporin-M” (strain 26D) on such indicators as the microbiological and chemical composition of leaves, the content of photosynthetic pigments in leaves, and chlorophyll fluorescence indices was studied. Plant samples were analyzed using standard methodology. When applying strains and microbiological preparations based on Bacillus subtilis, the taxonomic composition of endophytic bacterial forms cultured on dense nutrient media and isolated from leaves was represented by the Bacillus genus. For comparison, when treating plants with Bacillus subtilis strain V417 and “Phytosporin-M”, the Pseudomonas genus was also detected. The number of cultivated forms of endophytic bacteria in the treated plants was consistently growing, having reached the maximum value of 406800 CFU/g in the “Extrasol” variant. Notably, the content of photosynthetic pigments in leaves depended on both the plant variety and the strain used. High values of maximal efficiency of PSII photochemistry (Fv/Fm) were observed both in experimental and control variants. A consistent increase in leaf N content was detected when applying “Extrasol”. In this respect, the ‘President’ variety showed the greatest response, i.e., a 16.1 % increase compared to the control. The ‘President’ and ‘Triumph’ varieties demonstrated an increase in leaf K content under the influence of foliar treatment of plants with the studied strains and microbiological preparations. The greatest effect of 17.2 % above the control was observed in the ‘Triumph’ variety when treated with “Extrasol”. Foliar treatments of ‘President’ and ‘Valuta’ plants with microbiological preparations contributed to increased, compared to the control, Mg contents in their leaves.

Changes in reactive oxygen species and autofluorescence under hypoxia at the hippocampal CA3 area: Role of calcium and zinc influxes

Reactive oxygen species (ROS) have an important role in cellular biology, being involved, in a way that depends on their levels, in cell signaling processes or in oxidative stress, probably associated with neurodegenerative and other diseases. Most of the studies about ROS formation were performed in ischemic conditions, and thus, there is limited knowledge about ROS formation in less severe hypoxic conditions. This study investigates neuronal ROS generation and autofluorescence changes in hypoxic conditions, focusing on the involvement of calcium and zinc. Using hippocampal slices from Wistar rats, ROS production was monitored by the permeant fluorescent indicator H2DCFDA under different oxygenation levels. Moderate hypoxia (40% O2) led to a small ROS increase, while severe hypoxia (0% O2) showed a more pronounced rise. KCl-induced depolarization significantly enhanced ROS formation, particularly under severe hypoxia. Inhibition of NMDA receptors reduced ROS generation without affecting autofluorescence, while chelation of zinc ions decreased ROS production and increased flavin adenine dinucleotide (FAD) autofluorescence. These findings suggest that, in hypoxic conditions, ROS formation is mediated by calcium entry through NMDA receptors and also by zinc influxes. Thus, these ions play a crucial role in oxidative stress, which may be related with neurodegenerative diseases associated with ROS dysregulation.

Two-Photon Microscopy to Measure Calcium Signaling in the Living Brain.

Two-photon microscopy enables imaging of calcium signaling at cellular or subcellular resolution up to hundreds of microns deep in the living brain. Changes in the brightness of fluorescent calcium indicators provide a readout of calcium levels over time, affording information about neuronal activity and/or calcium-dependent subcellular signaling. Here, we describe a protocol for repeated two-photon imaging of calcium signals in mice expressing a genetically encoded calcium indicator that have been implanted with a chronic cranial window.

Using physicochemical properties to predict the impact of natural dissolved organic carbon on transepithelial potential in the freshwater rainbow trout (Oncorhynchus mykiss) at neutral and acidic pH.

Dissolved organic carbon (DOC) is a complex mixture of molecules that varies in composition based on origin as well as spatial and temporal factors. DOC is an important water quality parameter as it regulates many biological processes in freshwater systems, including the physiological function of the gills in fish. These effects are often beneficial, especially at low pH where DOCs mitigate ion loss and protect active ion uptake. DOCs of different compositions and quality have varied ionoregulatory effects. The molecular variability of DOCs can be characterized using optical and chemical indices, but how these indices relate to the physiological effects exerted by DOCs is not well understood. We tested the effects of five naturally sourced DOCs, at both pH 7 and pH 4, on transepithelial potential (TEP) (a diffusion potential between the blood plasma and the external water) in rainbow trout. The five chosen DOCs have been well characterized and span large differences in physicochemical characteristics. Each of the DOCs significantly influenced TEP, although in a unique manner or magnitude which was likely due to their physicochemical characteristics. These TEP responses were also a function of pH. With the goal of determining which physicochemical indices are predictive of changes in TEP, we evaluated correlations between various indices and TEP at pH 7 and pH 4. The indices included: specific absorbance coefficient at 340nm, molecular weight index, fluorescence index, octanol-water partition coefficient, molecular charge, proton binding index, % humic acid-like, % fulvic acid-like, and % protein-like components by parallel factor analysis on fluorescence data (PARAFAC). Our results demonstrate the novel finding that there are three particularly important indices that are predictors of changes in TEP across pHs in rainbow trout: specific absorbance coefficient at 340nm, octanol-water partition coefficient; and proton binding index.

Resistance artery vasodilator pathways involved in the antihypertensive effects of cocoa shell extract in rats exposed to fetal undernutrition.

Fetal undernutrition establishes the foundations for hypertension development, with oxidative stress being a key hallmark. A growing interest in nutraceuticals for treating hypertension and environmental waste concerns prompted the present study aiming to evaluate whether supplementation with a polyphenol enriched extract from cocoa shell (CSE), a by-product from the chocolate industry with antioxidant properties, reduces hypertension of developmental origin, thus improving mesenteric resistance artery (MRA) vasodilatation. Adult male and female offspring from rats exposed to 50% food restriction from mid-gestation (maternal undernutrition, MUN) and controls were used. Supplementation was given through a gelatine (vehicle, VEH) or containing CSE (250mgkg-1day-1) 5daysweek-1 for 3weeks. Systolic blood pressure (SBP) was assessed by tail-cuff plethysmography. MRA function was studied by wire myography, and superoxide anion and nitric oxide were investigated by fluorescent indicators and confocal microscopy. Compared to control-VEH, MUN-VEH males showed significantly higher SBP, reduced MRA as well as relaxation to ACh, sodium nitroprusside and the AMPK agonist 5-aminoimidazole-4-carboxamide riboside, but not to isoproterenol. In MUN males, endothelial endothelium-derived hyperpolarizing factor and nitric oxide were unaltered, but MRA released a vasoconstrictor prostanoid and produced higher levels of superoxide anion. CSE normalized blood pressure and improved all above-mentioned MRA alterations in MUN males without an effect on control counterparts, except the reduction of superoxide anion. MUN-VEH females were normotensive and only showed a tendency towards larger superoxide anion production, which was abolished by CSE. CSE supplementation reduces SBP improving endothelium-dependent and independent MRA vasodilatation, related to local superoxide anion reduction, being a potential nutraceutical ingredient to counteract hypertension, in addition to contributing to the circular economy. KEY POINTS: Fetal undernutrition induces hypertension in males associated with deficient resistance artery vasodilatation, being normalized by cocoa shell extract (CSE). Release of a cyclooxygenase-derived contractile factor is the main endothelial alteration, which is abolished by CSE. AMPK and soluble guanylyl cyclase-mediated relaxation are also reduced in smooth muscle cells from maternal undernutrition resistance arteries, being improved by CSE. Vascular oxidative damage caused by excess superoxide anion generation can account for impaired vasodilatation, which is improved by CSE. The capacity of CSE to improve relaxation is probably related to its antioxidant bioactive factors, and thus cocoa shell is a potential food by-product to treat hypertension.

Effects of subsurface drip fertigation on potato growth, yield, and soil moisture dynamics

AimsThis study aimed to evaluate the impact of subsurface drip fertigation (SDF) on soil moisture content, potato growth, and tuber yield in loam soils, and compare these results with conventional surface drip fertigation (CF). The focus was on determining whether SDF could improve water use efficiency and yield quality, particularly in water-scarce regions.MethodsThe experiment was conducted during the 2022 spring growing season in Xunyang, Ankang, Shaanxi Province, China. A randomized complete block design (RCBD) was used with three treatments: subsurface drip fertigation (SDF), conventional surface drip fertigation (CF), and a no-fertilization control (NF), with four replications per treatment. Soil moisture content at a 20 cm depth was monitored, and plant growth parameters such as plant height, stem diameter, leaf color index, and chlorophyll fluorescence index were measured during the flowering and harvest stages. Tuber yield characteristics, including tuber diameter, number of tubers per plant, total yield, and marketable yield, were also assessed.Important findingsThe results indicated that subsurface drip fertigation significantly improved soil moisture content, with up to 45.5% higher moisture retention compared to conventional fertigation, particularly in the early stages of fertilization. This improved moisture availability led to enhanced plant growth and tuber development. Tuber diameter increased by 6.9 mm, and the number of tubers per plant increased by 18.1% under SDF. Marketable tuber yield was approximately 10% higher in the SDF treatment compared to CF. However, the study found that soil texture plays a critical role in the effectiveness of SDF, and further research is needed to explore its application in other soil types.

Ex vivo propagation of synaptically-evoked cortical depolarizations in a mouse model of Alzheimer’s disease at 20 Hz, 40 Hz, or 83 Hz

Sensory stimulations at 40 Hz gamma (but not any other frequency), have shown promise in reversing Alzheimer’s disease (AD)-related pathologies. What distinguishes 40 Hz? We hypothesized that stimuli at 40 Hz might summate more efficiently (temporal summation) or propagate more efficiently between cortical layers (vertically), or along cortical laminas (horizontally), compared to inputs at 20 or 83 Hz. To investigate these hypotheses, we used brain slices from AD mouse model animals (5xFAD). Extracellular (synaptic) stimuli were delivered in cortical layer 4 (L4). Leveraging a fluorescent voltage indicator (VSFP) expressed in cortical pyramidal neurons, we simultaneously monitored evoked cortical depolarizations at multiple sites, at 1 kHz sampling frequency. Experimental groups (AD-Female, CTRL-Female, AD-Male, and CTRL-Male) were tested at three stimulation frequencies (20, 40, and 83 Hz). Despite our initial hypothesis, two parameters—temporal summation of voltage waveforms and the strength of propagation through the cortical neuropil—did not reveal any distinct advantage of 40 Hz stimulation. Significant physiological differences between AD and Control mice were found at all stimulation frequencies tested, while the 40 Hz stimulation frequency was not remarkable.

HCN1 hyperpolarization-activated cyclic nucleotide–gated channels enhance evoked GABA release from parvalbumin-positive interneurons

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels generate the cationic Ih current in neurons and regulate the excitability of neuronal networks. The function of HCN channels depends, in part, on their subcellular localization. Of the four HCN isoforms (HCN1-4), HCN1 is strongly expressed in the dendrites of pyramidal neurons (PNs) in hippocampal area CA1 but also in presynaptic terminals of parvalbumin-positive interneurons (PV+ INs), which provide strong inhibitory control over hippocampal activity. Yet, little is known about how HCN1 channels in these cells regulate the evoked release of the inhibitory transmitter GABA from their axon terminals. Here, we used genetic, optogenetic, electrophysiological, and imaging techniques to investigate how the electrophysiological properties of PV+ INs are regulated by HCN1, including how HCN1 activity at presynaptic terminals regulates the release of GABA onto PNs in CA1. We found that application of HCN1 pharmacological blockers reduced the amplitude of the inhibitory postsynaptic potential recorded from CA1 PNs in response to selective optogenetic stimulation of PV+ INs. Homozygous HCN1 knockout mice also show reduced IPSCs in postsynaptic cells. Finally, two-photon imaging using genetically encoded fluorescent calcium indicators revealed that HCN1 blockers reduced the probability that an extracellular electrical stimulating pulse evoked a Ca2+ response in individual PV+ IN presynaptic boutons. Taken together, our results show that HCN1 channels in the axon terminals of PV+ interneurons facilitate GABAergic transmission in the hippocampal CA1 region.

9291 Hypothyroidism impairs skeletal muscle regeneration after injury

Abstract Disclosure: P. Aguiari: None. V. Villani: None. K.Y. Liu: None. G.A. Brent: None. L. Perin: None. A. Milanesi: None. Thyroid hormone (TH) signaling plays an essential role in muscle development and function, in the maintenance of muscle mass, and in regeneration after injury. Disruption of TH signaling results in an abnormal skeletal muscle phenotype, impaired regeneration, and sarcopenia with aging, reflecting the myopathic changes described in hypothyroid patients. Muscle stem cells (MuSCs) are the mediators of post-natal skeletal muscle plasticity. Normally quiescent, in response to injury they enter the cell cycle (myoblasts) and proliferate. Myoblasts then exit the cell cycle and differentiate into myocytes that will fuse with existing myofibers to restore tissue architecture and function. Via TH receptor alpha, TH regulates all the stages of the regeneration program and regulates the expression of multiple myogenic genes. Despite all this evidence, to date there are no studies investigating MuSCs behavior in response to injury during hypothyroidism. We characterized injury-induced regeneration of the tibialis anterior muscle (TAM) in euthyroid and hypothyroid mice, fed a low iodine + 0.15% propylthiouracil diet. To investigate the cell cycle dynamics of MuSCs, we generated Pax7-Fucci mice carrying the fluorescent ubiquitination-based cell-cycle indicator (Fucci) system under the Pax7 promoter. Muscle injury was induced via cardiotoxin injection in the TAM of 3-month-old Pax7-Fucci mice. Hypothyroid mice present signs of impaired regeneration compared to euthyroid mice. From histological analysis, at different time points, we observed smaller fiber diameters, myofibers with central nuclei, and a significantly reduced number of fast glycolytic type IIb fibers, the prevalent muscle fiber type in the TA muscle. ScRNAseq analysis shows that at a late phase of regeneration (14 days after injury) hypothyroid MuSCs and myoblasts are still in the activation state and overexpress genes related to DNA replication and cell proliferation, while genes related to myogenic differentiation and cell cycle exit are downregulated. Differentiated myocytes in the hypothyroid fibers present an immature phenotype and are characterized by overexpression of embryonic/temporary and slow myosin genes and downregulation of mature fast myosins. Cell cycle analysis of the Fucci signal through Flow Cytometry confirmed a higher number of activated hypothyroid MuSCs at 4, 7, and 28 days after injury. These cells accumulate in the S phase and are unable to exit the cell cycle and differentiate towards myocytes. These data demonstrate that hypothyroidism impairs muscle tissue regeneration halting MuSCs progression through the cell cycle, myoblast cell cycle exit, and fiber maturation. Investigating muscle stem cell behavior and response to injury during hypothyroidism is crucial to understanding the mechanism behind thyroid hormone-induced myopathies and identifying possible therapeutical targets. Presentation: 6/2/2024

8670 Hypothyroidism impairs skeletal muscle regeneration after injury

Abstract Disclosure: P. Aguiari: None. V. Villani: None. K.Y. Liu: None. G.A. Brent: None. L. Perin: None. A. Milanesi: None. Thyroid hormone (TH) signaling plays an essential role in muscle development and function, in the maintenance of muscle mass, and in regeneration after injury. Disruption of TH signaling results in an abnormal skeletal muscle phenotype, impaired regeneration, and sarcopenia with aging, reflecting the myopathic changes described in hypothyroid patients. Muscle stem cells (MuSCs) are the mediators of post-natal skeletal muscle plasticity. Normally quiescent, in response to injury they enter the cell cycle (myoblasts) and proliferate. Myoblasts then exit the cell cycle and differentiate into myocytes that will fuse with existing myofibers to restore tissue architecture and function. Via TH receptor alpha, TH regulates all the stages of the regeneration program and regulates the expression of multiple myogenic genes. Despite all this evidence, to date there are no studies investigating MuSCs behavior in response to injury during hypothyroidism. We characterized injury-induced regeneration of the tibialis anterior muscle (TAM) in euthyroid and hypothyroid mice, fed a low iodine + 0.15% propylthiouracil diet. To investigate the cell cycle dynamics of MuSCs, we generated Pax7-Fucci mice carrying the fluorescent ubiquitination-based cell-cycle indicator (Fucci) system under the Pax7 promoter. Muscle injury was induced via cardiotoxin injection in the TAM of 3-month-old Pax7-Fucci mice. Hypothyroid mice present signs of impaired regeneration compared to euthyroid mice. From histological analysis, at different time points, we observed smaller fiber diameters, myofibers with central nuclei, and a significantly reduced number of fast glycolytic type IIb fibers, the prevalent muscle fiber type in the TA muscle. ScRNAseq analysis shows that at a late phase of regeneration (14 days after injury) hypothyroid MuSCs and myoblasts are still in the activation state and overexpress genes related to DNA replication and cell proliferation, while genes related to myogenic differentiation and cell cycle exit are downregulated. Differentiated myocytes in the hypothyroid fibers present an immature phenotype and are characterized by overexpression of embryonic/temporary and slow myosin genes and downregulation of mature fast myosins. Cell cycle analysis of the Fucci signal through Flow Cytometry confirmed a higher number of activated hypothyroid MuSCs at 4, 7, and 28 days after injury. These cells accumulate in the S phase and are unable to exit the cell cycle and differentiate towards myocytes. These data demonstrate that hypothyroidism impairs muscle tissue regeneration halting MuSCs progression through the cell cycle, myoblast cell cycle exit, and fiber maturation. Investigating muscle stem cell behavior and response to injury during hypothyroidism is crucial to understanding the mechanism behind thyroid hormone-induced myopathies and identifying possible therapeutical targets. Presentation: 6/2/2024

Long-term rice-crayfish farming alters soil dissolved organic carbon quality and biodegradability by regulating microbial metabolism and iron oxidation

The biodegradability of dissolved organic carbon (DOC) is a crucial process in the migration and transformation of soil organic carbon (SOC), and play a vital role in the global soil carbon (C) cycle. Although the significance of DOC in SOC transportation and microbial utilization is widely acknowledged, the impact of long-term rice-crayfish (RC) farming on the content, quality, and biodegradability of DOC in paddy soils, as well as regulatory mechanisms involved, remains unclear. To address this gap, a space-for-time method was employed to investigate the effects of different RC farming durations (1-, 5-, 10-, 15-, and 20- years) on the quality and biodegradability of DOC, as well as their relationship with soil microbial metabolism and minerals in this study. The results revealed that continuous RC farming increased the soil DOC content, but reduced DOC biodegradability. Specifically, after 20 years of continuous RC farming, the DOC content increased by 52.7% compared to the initial year, whereas the DOC biodegradability decreased by 63.4%. Analysis using three-dimensional fluorescence and ultraviolet spectroscopy demonstrated that continuous RC farming resulted in a decrease in the relative abundance of humus-like fractions, humification, and aromaticity indexes in DOC, but increased the relative abundance of protein-like fractions, biological, and fluorescence index, indicating that long-term RC farming promoted the simple depolymerization of the molecular structure of DOC. Continuous RC farming increased the activity of hydrolase involved in soil nitrogen (N) and phosphorus (P) cycles and oxidase, but decreasing the hydrolase C/N and C/P acquisition ratios; moreover, it also stimulated an increase in soil iron oxides and exchangeable calcium content. Structural equation modeling suggests that soil hydrolases and iron oxides are the primary drivers of DOC quality change, with DOC biodegradability being driven solely by soil iron oxides and not regulated by DOC quality. In conclusion, long-term RC farming promotes the catalytic decomposition of DOC aromatic substances and the production of DOC protein-like components by increasing soil oxidase activity and decreasing the hydrolase C/N acquisition ratio; these processes collectively contribute to the simple depolymerization of DOC molecular structure. Additionally, long-term RC farming induced legacy effects of soil iron oxides and enhanced chemical protection role leading to reduced DOC biodegradability. These findings suggested that long-term RC farming may reduce the rapid turnover and loss of DOC, providing a negative feedback on climate warming.