FDX1-dependent and independent mechanisms of elesclomol-mediated intracellular copper delivery.

Recent studies have uncovered the therapeutic potential of elesclomol (ES), a copper-ionophore, for copper deficiency disorders. However, we currently do not understand the mechanism by which copper brought into cells as ES-Cu(II) is released and delivered to cuproenzymes present in different subcellular compartments. Here, we have utilized a combination of genetic, biochemical, and cell-biological approaches to demonstrate that intracellular release of copper from ES occurs inside and outside of mitochondria. The mitochondrial matrix reductase, FDX1, catalyzes the reduction of ES-Cu(II) to Cu(I), releasing it into mitochondria where it is bioavailable for the metalation of mitochondrial cuproenzyme- cytochrome c oxidase. Consistently, ES fails to rescue cytochrome c oxidase abundance and activity in copper-deficient cells lacking FDX1. In the absence of FDX1, the ES-dependent increase in cellular copper is attenuated but not abolished. Thus, ES-mediated copper delivery to nonmitochondrial cuproproteins continues even in the absence of FDX1, suggesting alternate mechanism(s) of copper release. Importantly, we demonstrate that this mechanism of copper transport by ES is distinct from other clinically used copper-transporting drugs. Our study uncovers a unique mode of intracellular copper delivery by ES and may further aid in repurposing this anticancer drug for copper deficiency disorders.

Transplantation of beige adipose organoids fabricated using adipose acellular matrix hydrogel improves metabolic dysfunction in high-fat diet-induced obesity and type 2 diabetes mice.

Transplantation of brown adipose tissue (BAT) is a promising approach for treating obesity and metabolic disorders. However, obtaining sufficient amounts of functional BAT or brown adipocytes for transplantation remains a major challenge. In this study, we developed a hydrogel that combining adipose acellular matrix (AAM) and GelMA and HAMA that can be adjusted for stiffness by modulating the duration of light-crosslinking. We used human white adipose tissue-derived microvascular fragments to create beige adipose organoids (BAO) that were encapsulated in either a soft or stiff AAM hydrogel. We found that BAOs cultivated in AAM hydrogels with high stiffness demonstrated increased metabolic activity and upregulation of thermogenesis-related genes. When transplanted into obese and type 2 diabetes mice, the HFD + BAO group showed sustained improvements in metabolic rate, resulting in significant weight loss and decreased blood glucose levels. Furthermore, the mice showed a marked reduction in nonalcoholic liver steatosis, indicating improved liver function. In contrast, transplantation of 2D-cultured beige adipocytes failed to produce these beneficial effects. Our findings demonstrate the feasibility of fabricating beige adipose organoids in vitro and administering them by injection, which may represent a promising therapeutic approach for obesity and diabetes.

GANSamples-ac4C: Enhancing ac4C site prediction via generative adversarial networks and transfer learning.

RNA modification, N4-acetylcytidine (ac4C), is enzymatically catalyzed by N-acetyltransferase 10 (NAT10) and plays an essential role across tRNA, rRNA, and mRNA. It influences various cellular functions, including mRNA stability and rRNA biosynthesis. Wet-lab detection of ac4C modification sites is highly resource-intensive and costly. Therefore, various machine learning and deep learning techniques have been employed for computational detection of ac4C modification sites. The known ac4C modification sites are limited for training an accurate and stable prediction model. This study introduces GANSamples-ac4C, a novel framework that synergizes transfer learning and generative adversarial network (GAN) to generate synthetic RNA sequences to train a better ac4C modification site prediction model. Comparative analysis reveals that GANSamples-ac4C outperforms existing state-of-the-art methods in identifying ac4C sites. Moreover, our result underscores the potential of synthetic data in mitigating the issue of data scarcity for biological sequence prediction tasks. Another major advantage of GANSamples-ac4C is its interpretable decision logic. Multi-faceted interpretability analyses detect key regions in the ac4C sequences influencing the discriminating decision between positive and negative samples, a pronounced enrichment of G in this region, and ac4C-associated motifs. These findings may offer novel insights for ac4C research. The GANSamples-ac4C framework and its source code are publicly accessible at http://www.healthinformaticslab.org/supp/.

Transplantation of in vitro prefabricated adipose organoids attenuates skin fibrosis by restoring subcutaneous fat and inducing dermal adipogenesis.

Localized scleroderma is a complex autoimmune disease characterized by dermal fibrosis and loss of cutaneous fat. While cytotherapy offers a promising treatment option, stem cell transplantation results in low survival rates and fails in target cell differentiation. In this study, we aimed to prefabricate syngeneic adipose organoids (ad-organoids) using microvascular fragments (MVFs) via three-dimensional (3D) culturing and transplant them beneath the fibrotic skin to restore subcutaneous fat and reverse the pathological manifestation of localized scleroderma. We employed 3D culturing of syngeneic MVFs with stepwise angiogenic and adipogenic induction to produce ad-organoids and evaluated their microstructure and paracrine function in vitro. C57/BL6 mice with induced skin scleroderma were treated with adipose-derived stem cells (ASCs), adipocytes, ad-organoids, and Matrigel, and the therapeutic effect was assessed histologically. Our results showed that ad-organoids derived from MVF contained mature adipocytes and a well-established vessel network, secreted multiple adipokines, promoted adipogenic differentiation of ASCs, and suppressed proliferation and migration of scleroderma fibroblasts. Subcutaneous transplantation of ad-organoids reconstructed the subcutaneous fat layer and stimulated dermal adipocyte regeneration in bleomycin-induced scleroderma skin. It reduced collagen deposition and dermal thickness, attenuating dermal fibrosis. Moreover, ad-organoids suppressed macrophage infiltration and promoted angiogenesis in the skin lesion. In conclusion, 3D culturing of MVFs with stepwise angiogenic and adipogenic induction is an effective strategy for the fabrication of ad-organoids, and the transplantation of prefabricated ad-organoids can improve skin sclerosis by restoring cutaneous fat and attenuating skin fibrosis. These findings offer a promising therapeutic approach for the treatment of localized scleroderma.

Turn-On Fluorescent Probe for BSA Detection Constructed by Supramolecular Assembly.

The fluorescent probe method has attracted significant research attention due to its high sensitivity and reproducibility in detecting bovine serum albumin (BSA). In this study, we constructed a fluorescent probe for BSA detection by assembling an amphiphilic organic fluorescent molecule, termed 2-(2'-hydroxyphenyl) benzothiazole (HBT-11), with BSA. In an aqueous solution, HBT-11 exhibited a weak fluorescence emission at 501 nm. However, the addition of BSA substantially enhanced the fluorescence emission at 501 nm, indicating that the assembly was driven by electrostatic interactions between HBT-11 and BSA. HBT-11, serving as a fluorescent probe for BSA detection, demonstrated a limit of detection (LOD) as low as 3.92 nmol L-1, excellent photostability, high selectivity, and robust anti-interference capability. Notably, we successfully applied HBT-11 for detecting BSA in fetal bovine serum and selectively imaging BSA in HeLa cells.

Exercise-induced gene expression changes in skeletal muscle of old mice.

Exercise is believed to be beneficial for skeletal muscle functions across all ages. Regimented exercise is often prescribed as an effective treatment/prophylaxis for age-related loss of muscle mass and function, known as sarcopenia, and plays an important role in the maintenance of mobility and functional independence in the elderly. However, response to exercise declines with aging, resulting in limited gain of muscle strength and endurance. These changes likely reflect age-dependent alterations in transcriptional response underlying the muscular adaptation to exercise. The exact changes in gene expression accompanying exercise, however, are largely unknown, and elucidating them is of a great clinical interest for understanding and optimizing the exercise-based therapies for sarcopenia. In order to characterize the exercise-induced transcriptomic changes in aged muscle, a paired-end RNA sequencing was performed on rRNA-depleted total RNA extracted from the gastrocnemius muscles of 24 months-old mice after 8 weeks of regimented exercise (exercise group) or no formal exercise program (sedentary group). Differential gene expression analysis of aged skeletal muscle revealed upregulations in the group of genes involved in neurotransmission and neuroexcitation, as well as equally notable absence of anabolic gene upregulations in the exercise group. In particular, genes encoding the transporters and receptor components of glutaminergic transmission were significantly upregulated in exercised muscles, as exemplified by Gria 1, Gria 2 and Grin2c encoding glutamate receptor 1, 2 and 2C respectively, Grin1 and Grin2b encoding N-methyl-d-aspartate receptors (NMDARs), Nptx1 responsible for glutaminergic receptor clustering, and Slc1a2 and Slc17a7 regulating synaptic uptake of glutamate. These changes were accompanied by an increase in the post-synaptic density of NMDARs and acetylcholine receptors (AChRs), as well as their innervation at neuromuscular junctions (NMJs). These results suggest that neural responses predominate the adaptive response of aged skeletal muscle to exercise, and indicate a possibility that glutaminergic transmission at NMJs may be present and responsible for synaptic protection and neural remodeling accompanying the exercise-induced functional enhancement in aged skeletal muscle. In addition, the absence of upregulations in the anabolic pathways highlights them as the area of potential pharmacological targeting for optimizing exercise-led sarcopenia therapy.

Loss of ARNT in skeletal muscle limits muscle regeneration in aging.

The ability of skeletal muscle to regenerate declines significantly with aging. The expression of aryl hydrocarbon receptor nuclear translocator (ARNT), a critical component of the hypoxia signaling pathway, was less abundant in skeletal muscle of old (23-25 months old) mice. This loss of ARNT was associated with decreased levels of Notch1 intracellular domain (N1ICD) and impaired regenerative response to injury in comparison to young (2-3 months old) mice. Knockdown of ARNT in a primary muscle cell line impaired differentiation in vitro. Skeletal muscle-specific ARNT deletion in young mice resulted in decreased levels of whole muscle N1ICD and limited muscle regeneration. Administration of a systemic hypoxia pathway activator (ML228), which simulates the actions of ARNT, rescued skeletal muscle regeneration in both old and ARNT-deleted mice. These results suggest that the loss of ARNT in skeletal muscle is partially responsible for diminished myogenic potential in aging and activation of hypoxia signaling holds promise for rescuing regenerative activity in old muscle.

Dissection of Interaction Kinetics through Single-Molecule Interaction Simulation.

The ability to extract kinetic interaction parameters from single-molecule fluorescence resonance energy transfer trajectories without the need for solving complex single-molecule differential equations has the potential to address some of the critical biophysical questions. Here, we provide a noise-free single-molecule interaction simulation (SMIS) tool to give the expected dwell-time distributions and relative populations of each FRET level based on the assigned kinetic model and to dissect kinetic interaction parameters from single-molecule FRET trajectories. The method provides the expected dwell-time distributions, average transition rates, and relative populations of each FRET level based on the assigned kinetic model. By comparing with ground truth data and experimental data, we demonstrated that SMIS is useful to quantify the interaction kinetic rate constants without using the traditional single-molecule analytical solution approach.

Face-Directed Assembly of Molecular Cubes: In Situ Substitution of a Predetermined Concave Cluster.

Systematic design and self-assembly of metal-organic polyhedra with predictable configurations has been a long-standing challenge in crystal engineering. Herein a concave polyoxovanadate cluster, [V6 O6 (OCH3 )9 (SO4 )4 ]5- , which can be generated in situ under specific reaction conditions, is reported. Based on this cluster, a potential trivalent molecular building block, [V6 O6 (OCH3 )9 (SO4 )(CO2 )3 ]2- , can be obtained by the bridging-ligand-substitution strategy and it possesses appropriate angle information for the design of molecular cubes. Utilizing the face-directed assembly of the trivalent molecular building block and a diverse set of tetratopic carboxylate linkers, a series of metal-organic cubes (VMOC-1-VMOC-5) with the same topology but different functionalities and dimensions were designed and constructed. An inclusion study using VMOC-3 shows that they are potential molecular receptors for selective capture of size-matching polycyclic aromatic hydrocarbon guest molecules.

Bottom-Up Construction and Reversible Structural Transformation of Supramolecular Isomers based on Large Truncated Tetrahedra.

A rational synthetic strategy to construct two supramolecular isomers based on polyoxovanadate organic polyhedra with tetrahedral symmetries is presented. VMOP-α, a low-temperature product, has an extremely large cell volume (470 842 Å3 ), which is one of the top three for well-defined MOPs. The corner-to-corner packing of tetrahedra leads to a quite low density of 0.174 g cm-3 with 1D channels (ca. 5.4 nm). The effective pore volume is up to 93.6 % of cell volume, nearly the largest found in MOPs. For the high-temperature outcome, VMOP-ß, the cell volume is only 15 513 Å3 . The packing mode of tetrahedra is corner-to-face, giving rise to a high-density architecture (1.324 g cm-3 ; channel 0.8 nm). Supramolecular structural transformation between VMOP-α and VMOP-ß can be reversibly achieved by temperature-induced solvent-mediated transformation. These findings give a good opportunity for understanding 3D supramolecular aggregation and crystal growth based on large molecular tectonics.

Self-Assembly of Goldberg Polyhedra from a Concave [WV5O11(RCO2)5(SO4)]3- Building Block with 5-Fold Symmetry.

Nanoscale regular polyhedra with icosahedral symmetry exist naturally as exemplified by virus capsids and fullerenes. Nevertheless, their generation by supramolecular chemistry through the linking of 5-fold symmetry vertices remains unmet because of the absence of 5-fold symmetry building blocks with the requisite geometric features. This situation contrasts with that of tetrahedral and octahedral symmetry metal-organic polyhedra (MOPs), for which appropriate triangular and square molecular building blocks (MBBs) that can serve as vertices or faces are readily available. Herein, we report isolation of a pentagonal [WV5O11(SO4)6]8- cluster and reveal its utility to afford the first four examples of nanoscale Goldberg MOPs, based upon 5-fold MBBs. Two 32-faced G v(1,1) MOPs and two 42-faced G v(2,0) MOPs were formed using linear or triangular organic ligands, respectively. The largest Goldberg MOP-4, exhibits a diameter of 4.3 nm, can trap fullerene C60 molecules in its interstitial cavities.

Ligand-Directed Assembly of Polyoxovanadate-Based Metal-Organic Polyhedra.

Three new polyoxovanadate-based metal-organic polyhedra (VMOPs) have been successfully synthesized and structurally characterized. Single crystals of three VMOPs were obtained by reaction of VCl3 and different carboxylate ligands (2,5-H2TDA = thiophene-2,5-dicarboxylic acid for VMOP-4, m-H2BDC = 1,3-benzenedicarboxylic acid for VMOP-5, 2,6-H2NDC = 2,6-naphthalenedicarboxylic aid for VMOP-6) under solvothermal conditions. Though all of the three hybrids feature the same {V(V)V4(IV)} units, their structures exhibit differences changing from truncated triangular prism to truncated quadrangular prism to octahedron, mainly depending on the nature of carboxylate ligands. Furthermore, the magnetic investigations reveal that VMOP-4-6 show similar ferromagnetic behaviors.

High-Nuclear Vanadoniobate {Nb48V8} Multiple-Strand Wheel.

An unprecedented octavanadium-substituted polyoxoniobate Na18[Nb48V8(OH)30O130] · 33H2O (1), with a multiple-strand wheel structure, was successfully synthesized via a conventional aqueous method, which represents the largest vanadoniobate cluster reported to date. Single-crystal X-ray diffraction, ESI-MS spectrum, IR spectra, and UV-vis spectra were investigated. In addition, photocatalytic H2 evolution activity for 1 under UV light was observed with TEA as a sacrificial electron donor.

Use of brown adipose tissue transplantation and engineering as a thermogenic therapy in obesity and metabolic disease.

The induction of thermogenesis in brown adipose tissue is emerging as an attractive therapy for obesity and metabolic syndrome. However, the long-term efficacy and safety of clinical pharmaceutical agents have yet to be fully characterized. The transplantation of brown adipose tissue represents an alternative approach that might have a therapeutic effect by inducing a long-term increase in energy expenditure. However, limited tissue resources hinder the development of transplantation. Stem cell-based therapy and brown adipose tissue engineering, in addition to transplantation, represent alternative approaches that might resolve this problem. In this article, we discuss recent advances in understanding the mechanisms and applications of brown adipose tissue transplantation in the treatment of obesity and related metabolic disorders. Specifically, the induction of brown adipocytes and the fabrication of engineered brown adipose tissue as novel transplantation resources have long-term effects on ameliorating metabolic defects in rodent models. Additionally, we explore future prospects regarding the development of three-dimensional engineered brown adipose tissue and the associated challenges.

Advancements and Practical Considerations for Biophysical Research: Navigating the Challenges and Future of Super-resolution Microscopy.

The introduction of super-resolution microscopy (SRM) has significantly advanced our understanding of cellular and molecular dynamics, offering a detailed view previously beyond our reach. Implementing SRM in biophysical research, however, presents numerous challenges. This review addresses the crucial aspects of utilizing SRM effectively, from selecting appropriate fluorophores and preparing samples to analyzing complex data sets. We explore recent technological advancements and methodological improvements that enhance the capabilities of SRM. Emphasizing the integration of SRM with other analytical methods, we aim to overcome inherent limitations and expand the scope of biological insights achievable. By providing a comprehensive guide for choosing the most suitable SRM methods based on specific research objectives, we aim to empower researchers to explore complex biological processes with enhanced precision and clarity, thereby advancing the frontiers of biophysical research.

Time series prediction of the chemical components of PM2.5 based on a deep learning model.

Modeling-based prediction methods enable rapid, reagent-free air pollution detection based on inexpensive multi-source data than traditional chemical reaction-based detection methods in order to quickly understand the air pollution situation. In this study, a convolutional neural network (CNN) and long and short-term memory (LSTM) neural networks are integrated to create a CNN-LSTM time series prediction model to predict the concentration of PM2.5 and its chemical components (i.e., heavy metals, carbon component, and water-soluble ions) using meteorological data and air pollutants (PM2.5, SO2, NO2, CO, and O3). In the integrated CNN-LSTM model, the CNN uses convolutional and pooling layers to extract features from the data, whereas the powerful nonlinear mapping and learning capabilities of LSTM enable the time series prediction of air pollution. The experimental results showed that the CNN-LSTM exhibited good generalization ability in the prediction of As, Cd, Cr, Cu, Ni, and Zn, with a mean R2 above 0.9. Mean R2 predicted for PM2.5, Pb, Ti, EC, OC, SO42-, and NO3- ranged from 0.85 to 0.9. Shapley value showed that PM2.5, NO2, SO2, and CO had a greater influence on the predicted heavy metal results of the model. Regarding water-soluble ions, the predicted results were dominantly influenced by PM2.5, CO, and humidity. The prediction of the carbon fraction was affected mainly by the PM2.5 concentration. Additionally, several input variables for various components were eliminated without affecting the prediction accuracy of the model, with R2 between 0.70 and 0.84, thereby maximizing modeling efficiency and lowering operational costs. The fully trained model prediction results showed that most predicted components of PM2.5 were lower during January to March 2020 than those in 2018 and 2019. This study provides insight into improving the accuracy of modeling-based detection methods and promotes the development of integrated air pollution monitoring toward a more sustainable direction.

Effects of the Chinese herbal formula San-Huang Gu-Ben Zhi-Ke treatment on stable chronic obstructive pulmonary disease: a randomized, double-blind, placebo-controlled trial.

Objective: The aim of this study was to evaluate the efficacy and safety of the Chinese herbal formula San-Huang Gu-Ben Zhi-Ke (SHGBZK) as a treatment for patients with stable chronic obstructive pulmonary disease (COPD) diagnosed with lung-spleen Qi deficiency. Method: A randomized, double-blind, placebo-controlled trial was designed. 98 adults aged between 40 and 80 years with stable COPD diagnosed with lung-spleen Qi deficiency were included. All participants received basic treatment for COPD. Patients in the experimental group took SHGBZK, while the control group took placebo. The primary outcome was the frequency of acute exacerbation. The secondary outcomes were lung function, symptom score, exercise capacity and quality of life. Results: Of 98 patients who underwent randomization, 50 patients in the SHGBZK group and 48 in the placebo group were included in the full analysis set. After 24-week therapy and 28-week follow-up, patients in treatment group had significant improvements in symptom, exercise capacity and quality of life. After Subgroup analysis, the frequency of acute exacerbation in patients with a COPD Assessment Test (CAT) score of at least 10 or a modified Medical Research Council (mMRC) score of at least 2 was significantly lower in the SHGBZK group than in the placebo group. Lung function in patients with frequent exacerbation was significantly higher in the SHGBZK group than in the placebo group. The incidence of adverse events was generally similar in the two groups. Conclusion: SHGBZK had beneficial effects on symptom, exercise capacity and quality of life in stable COPD patients. SHGBZK also had the potential to reduce the frequency of exacerbation and improve lung function in specific groups of COPD patients. Clinical Trial Registration: https://www.chictr.org.cn/showproj.html?proj=26933, identifier ChiCTR1800016349.

Inflammatory response in mouse lungs to haze episodes under different backgrounds of particulate matter exposure.

Particulate matter (PM) toxicity has mostly been investigated through in vitro exposure or tracheal infusion in animal models. However, given the complexity of ambient conditions, most animal studies do not mimic real-life PM exposure. In this work, we established a novel integrated exposure model to study the dynamic inflammatory response and defense strategies in ambient PM-exposed mice. Three groups of male C57BL/6 mice were kept in three chambers with pre-exposure to filtered air (FA), unfiltered air (UFA), or the air with a low PM concentration (PM2.5 ≤ 75 µg/m3) (LPM), respectively, for 37 days. Then all three groups of mice were exposed to haze challenge for 3 days, followed by exposure in filtered air for 7 days to allow recovery. Our results suggest that following a haze challenge, the defense strategies of mice of filtered air (FA) and low PM (LPM) groups comprised a form of "counterattack", whereas the response of the unfiltered air (UFA) group could be viewed as a "silence". While the latter strategy protected the lung tissues of mice from acute inflammatory damage, it also foreshadowed the development of chronic inflammatory diseases. These findings contribute to explaining previously documented PM-associated pathogenic mechanisms.

VEGFA Promotes Skeletal Muscle Regeneration in Aging.

Aging is associated with loss of skeletal muscle regeneration. Differentially regulated vascular endothelial growth factor (VEGF)A with aging may partially underlies this loss of regenerative capacity. To assess the role of VEGFA in muscle regeneration, young (12-14 weeks old) and old C57BL/6 mice (24,25 months old) are subjected to cryoinjury in the tibialis anterior (TA) muscle to induce muscle regeneration. The average cross-sectional area (CSA) of regenerating myofibers is 33% smaller in old as compared to young (p < 0.01) mice, which correlates with a two-fold loss of muscle VEGFA protein levels (p = 0.02). The capillary density in the TA is similar between the two groups. Young VEGFlo mice, with a 50% decrease in systemic VEGFA activity, exhibit a two-fold reduction in the average regenerating fiber CSA following cryoinjury (p < 0.01) in comparison to littermate controls. ML228, a hypoxia signaling activator known to increase VEGFA levels, augments muscle VEGFA levels and increases average CSA of regenerating fibers in both old mice (25% increase, p < 0.01) and VEGFlo (20% increase, p < 0.01) mice, but not in young or littermate controls. These results suggest that VEGFA may be a therapeutic target in age-related muscle loss.

Subcellular redox responses reveal different Cu-dependent antioxidant defenses between mitochondria and cytosol.

Excess intracellular Cu perturbs cellular redox balance and thus causes diseases. However, the relationship between cellular redox status and Cu homeostasis and how such an interplay is coordinated within cellular compartments has not yet been well established. Using combined approaches of organelle-specific redox sensor Grx1-roGFP2 and non-targeted proteomics, we investigate the real-time Cu-dependent antioxidant defenses of mitochondria and cytosol in live HEK293 cells. The Cu-dependent real-time imaging experiments show that CuCl2 treatment results in increased oxidative stress in both cytosol and mitochondria. In contrast, subsequent excess Cu removal by bathocuproine sulfonate, a Cu chelating reagent, lowers oxidative stress in mitochondria but causes even higher oxidative stress in the cytosol. The proteomic data reveal that several mitochondrial proteins, but not cytosolic ones, undergo significant abundance change under Cu treatments. The proteomic analysis also shows that proteins with significant changes are related to mitochondrial oxidative phosphorylation and glutathione synthesis. The differences in redox behaviors and protein profiles in different cellular compartments reveal distinct mitochondrial and cytosolic response mechanisms upon Cu-induced oxidative stress. These findings provide insights into how redox and Cu homeostasis interplay by modulating specific protein expressions at the subcellular levels, shedding light on understanding the effects of Cu-induced redox misregulation on the diseases.