An extra-erythrocyte role of haemoglobin body in chondrocyte hypoxia adaption.

Although haemoglobin is a known carrier of oxygen in erythrocytes that functions to transport oxygen over a long range, its physiological roles outside erythrocytes are largely elusive1,2. Here we found that chondrocytes produced massive amounts of haemoglobin to form eosin-positive bodies in their cytoplasm. The haemoglobin body (Hedy) is a membraneless condensate characterized by phase separation. Production of haemoglobin in chondrocytes is controlled by hypoxia and is dependent on KLF1 rather than the HIF1/2α pathway. Deletion of haemoglobin in chondrocytes leads to Hedy loss along with severe hypoxia, enhanced glycolysis and extensive cell death in the centre of cartilaginous tissue, which is attributed to the loss of the Hedy-controlled oxygen supply under hypoxic conditions. These results demonstrate an extra-erythrocyte role of haemoglobin in chondrocytes, and uncover a heretofore unrecognized mechanism in which chondrocytes survive a hypoxic environment through Hedy.

Apoptotic Vesicular Metabolism Contributes to Organelle Assembly and Safeguards Liver Homeostasis and Regeneration.

BACKGROUND & AIMS: Apoptosis generates plenty of membrane-bound nanovesicles, the apoptotic vesicles (apoVs), which show promise for biomedical applications. The liver serves as a significant organ for apoptotic material removal. Whether and how the liver metabolizes apoptotic vesicular products and contributes to liver health and disease is unrecognized. METHODS: apoVs were labeled and traced after intravenous infusion. Apoptosis-deficient mice by Fas mutant (Fasmut) and Caspase-3 knockout (Casp3-/-) were used with apoV replenishment to evaluate the physiological apoV function. Combinations of morphologic, biochemical, cellular, and molecular assays were applied to assess the liver while hepatocyte analysis was performed. Partial hepatectomy and acetaminophen liver failure models were established to investigate liver regeneration and disease recovery. RESULTS: We discovered that the liver is a major metabolic organ of circulatory apoVs, in which apoVs undergo endocytosis by hepatocytes via a sugar recognition system. Moreover, apoVs play an indispensable role to counteract hepatocellular injury and liver impairment in apoptosis-deficient mice upon replenishment. Surprisingly, apoVs form a chimeric organelle complex with the hepatocyte Golgi apparatus through the soluble N-ethylmaleimide-sensitive factor attachment protein receptor machinery, which preserves Golgi integrity, promotes microtubule acetylation by regulating α-tubulin N-acetyltransferase 1, and consequently facilitates hepatocyte cytokinesis for liver recovery. The assembly of the apoV-Golgi complex is further revealed to contribute to liver homeostasis, regeneration, and protection against acute liver failure. CONCLUSIONS: These findings establish a previously unrecognized functional and mechanistic framework that apoptosis through vesicular metabolism safeguards liver homeostasis and regeneration, which holds promise for hepatic disease therapeutics.

Chronic intermittent hypoxia alters the dendritic mitochondrial structure and activity in the pre-Bötzinger complex of rats.

Mitochondrial bioenergetics is dynamically coupled with neuronal activities, which are altered by hypoxia-induced respiratory neuroplasticity. Here we report structural features of postsynaptic mitochondria in the pre-Bötzinger complex (pre-BötC) of rats treated with chronic intermittent hypoxia (CIH) simulating a severe condition of obstructive sleep apnea. The subcellular changes in dendritic mitochondria and histochemistry of cytochrome c oxidase (CO) activity were examined in pre-BötC neurons localized by immunoreactivity of neurokinin 1 receptors. Assays of mitochondrial electron transport chain (ETC) complex I, IV, V activities, and membrane potential were performed in the ventrolateral medulla containing the pre-BötC region. We found significant decreases in the mean length and area of dendritic mitochondria in the pre-BötC of CIH rats, when compared to the normoxic control and hypoxic group with daily acute intermittent hypoxia (dAIH) that evokes robust synaptic plasticity. Notably, these morphological alterations were mainly observed in the mitochondria in close proximity to the synapses. In addition, the proportion of mitochondria presented with enlarged compartments and filamentous cytoskeletal elements in the CIH group was less than the control and dAIH groups. Intriguingly, these distinct characteristics of structural adaptability were observed in the mitochondria within spatially restricted dendritic spines. Furthermore, the proportion of moderately to darkly CO-reactive mitochondria was reduced in the CIH group, indicating reduced mitochondrial activity. Consistently, mitochondrial ETC enzyme activities and membrane potential were lowered in the CIH group. These findings suggest that hypoxia-induced respiratory plasticity was characterized by spatially confined mitochondrial alterations within postsynaptic spines in the pre-BötC neurons. In contrast to the robust plasticity evoked by dAIH preconditioning, a severe CIH challenge may weaken the local mitochondrial bioenergetics that the fuel postsynaptic activities of the respiratory motor drive.

[Chronic intermittent hypoxia induces expression of phospho-PKC substrates in rat pre-Bötzinger complex].

The pre-Bötzinger complex (pre-BötC) residing in the ventrolateral medulla oblongata, is thought to be the kernel of respiratory rhythmogenesis. Episodic hypoxia exerts respiratory long-term facilitation, being recognized as electrophysiological characteristic of respiratory motor neuroplasticity. Our previous study demonstrated up-regulated expression of phospho-protein kinase C θ (P-PKCθ) in the pre-BötC of rats receiving chronic intermittent hypoxic (CIH) challenge. The present study was aimed to examine subcellular distribution of P-PKC substrates (P-PKCsub) and explore PKC down-stream targeting proteins in the pre-BötC in normoxic and CIH rats. Using neurokinin-1 receptor (NK1R) as a marker of the pre-BötC, P-PKCsub immunoreactivity was revealed by immunofluorescence and immuno-electron microscopic double-labeling in the pre-BötC. Western blot was applied to analyze P-PKCsub proteins in ventrolateral medulla, containing the pre-BötC. The results showed that NK1R immunoreactivity (NK1R-ir) was expressed mainly along plasma membranes of somata and processes, outlining pre-BötC neurons under the light microscope. P-PKCsub immunoreactive (P-PKCsub-ir) fluorophores in dot-like appearance appeared in somata and processes. Some were in close apposition to plasma membranes. A majority of P-PKCsub-ir neurons was found with NK1R-ir. CIH challenge up-regulated the expression of P-PKCsub proteins in the ventrolateral medulla. Under the electron microscope, NK1R-ir product was found to distribute along the inner membrane surfaces of somata and dendrites. P-PKCsub-ir gold particles were located in somata and dendrites, and some were distributed along the inner membrane surfaces, as well as in the endoplasmic reticulum and postsynaptic dense body. These results suggest that CIH challenge up-regulates the expression of P-PKCsub proteins, probably including some receptor proteins in the postsynaptic membrane, which may contribute to respiratory neuroplasticity via activation of PKCθ in the pre-BötC.

Electrochemical sensors based on molecularly imprinted polymers on Fe3O4/graphene modified by gold nanoparticles for highly selective and sensitive detection of trace ractopamine in water.

A novel molecular imprinting polymer (MIP)-based electrochemical senor, consisting of Fe3O4 nanobeads and gold nanoparticles on a reduced graphene oxide (RGO) substrate, was fabricated to detect ractopamine (RAC) in water using the reversible addition fragmentation chain transfer (RAFT) polymerization technique. The Au nanoparticles widely dispersed on RGO can significantly increase the response current for RAC detection in water, which is confirmed by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and theoretical calculations. By means of the differential pulse voltammetry technique, the as-prepared MIP-based electrode shows a dynamic linear range of 0.002 to 0.1 µM with a correlation coefficient of 0.992 and a remarkably low detection limit of 0.02 nM (S/N = 3). Additionally, the sensor exhibits high binding affinity and selectivity towards RAC with excellent reproducibility. Our study demonstrates the potential for the proposed electrochemical sensors in monitoring organic pollutants in water.

CB1R dysfunction of inhibitory synapses in the ACC drives chronic social isolation stress-induced social impairments in male mice.

Social isolation is a risk factor for multiple mood disorders. Specifically, social isolation can remodel the brain, causing behavioral abnormalities, including sociability impairments. Here, we investigated social behavior impairment in mice following chronic social isolation stress (CSIS) and conducted a screening of susceptible brain regions using functional readouts. CSIS enhanced synaptic inhibition in the anterior cingulate cortex (ACC), particularly at inhibitory synapses of cholecystokinin (CCK)-expressing interneurons. This enhanced synaptic inhibition in the ACC was characterized by CSIS-induced loss of presynaptic cannabinoid type-1 receptors (CB1Rs), resulting in excessive axonal calcium influx. Activation of CCK-expressing interneurons or conditional knockdown of CB1R expression in CCK-expressing interneurons specifically reproduced social impairment. In contrast, optogenetic activation of CB1R or administration of CB1R agonists restored sociability in CSIS mice. These results suggest that the CB1R may be an effective therapeutic target for preventing CSIS-induced social impairments by restoring synaptic inhibition in the ACC.

Calcified apoptotic vesicles from PROCR+ fibroblasts initiate heterotopic ossification.

Heterotopic ossification (HO) comprises the abnormal formation of ectopic bone in extraskeletal soft tissue. The factors that initiate HO remain elusive. Herein, we found that calcified apoptotic vesicles (apoVs) led to increased calcification and stiffness of tendon extracellular matrix (ECM), which initiated M2 macrophage polarization and HO progression. Specifically, single-cell transcriptome analyses of different stages of HO revealed that calcified apoVs were primarily secreted by a PROCR+ fibroblast population. In addition, calcified apoVs enriched calcium by annexin channels, absorbed to collagen I via electrostatic interaction, and aggregated to produce calcifying nodules in the ECM, leading to tendon calcification and stiffening. More importantly, apoV-releasing inhibition or macrophage deletion both successfully reversed HO development. Thus, we are the first to identify calcified apoVs from PROCR+ fibroblasts as the initiating factor of HO, and might serve as the therapeutic target for inhibiting pathological calcification.

Spatial memory requires hypocretins to elevate medial entorhinal gamma oscillations.

The hypocretin (Hcrt) (also known as orexin) neuropeptidic wakefulness-promoting system is implicated in the regulation of spatial memory, but its specific role and mechanisms remain poorly understood. In this study, we revealed the innervation of the medial entorhinal cortex (MEC) by Hcrt neurons in mice. Using the genetically encoded G-protein-coupled receptor activation-based Hcrt sensor, we observed a significant increase in Hcrt levels in the MEC during novel object-place exploration. We identified the function of Hcrt at presynaptic glutamatergic terminals, where it recruits fast-spiking parvalbumin-positive neurons and promotes gamma oscillations. Bidirectional manipulations of Hcrt neurons' projections from the lateral hypothalamus (LHHcrt) to MEC revealed the essential role of this pathway in regulating object-place memory encoding, but not recall, through the modulation of gamma oscillations. Our findings highlight the significance of the LHHcrt-MEC circuitry in supporting spatial memory and reveal a unique neural basis for the hypothalamic regulation of spatial memory.

Expression of phospho-Ca(2+) /calmodulin-dependent protein kinase II in the pre-Bötzinger complex of rats.

The pre-Bötzinger complex (pre-BötC) in the ventrolateral medulla oblongata is a presumed kernel of respiratory rhythmogenesis. Ca(2+) -activated non-selective cationic current is an essential cellular mechanism for shaping inspiratory drive potentials. Ca(2+) /calmodulin-dependent protein kinase II (CaMKII), an ideal 'interpreter' of diverse Ca(2+) signals, is highly expressed in neurons in mediating various physiological processes. Yet, less is known about CaMKII activity in the pre-BötC. Using neurokinin-1 receptor as a marker of the pre-BötC, we examined phospho (P)-CaMKII subcellular distribution, and found that P-CaMKII was extensively expressed in the region. P-CaMKII-ir neurons were usually oval, fusiform, or pyramidal in shape. P-CaMKII immunoreactivity was distributed within somas and dendrites, and specifically in association with the post-synaptic density. In dendrites, most synapses (93.1%) examined with P-CaMKII expression were of asymmetric type, occasionally with symmetric type (6.9%), whereas in somas, 38.1% were of symmetric type. P-CaMKII asymmetric synaptic identification implicates that CaMKII may sense and monitor Ca(2+) activity, and phosphorylate post-synaptic proteins to modulate excitatory synaptic transmission, which may contribute to respiratory modulation and plasticity. In somas, CaMKII acts on both symmetric and asymmetric synapses, mediating excitatory and inhibitory synaptic transmission. P-CaMKII was also localized to the perisynaptic and extrasynaptic regions in the pre-BötC.

Alterations in synapses and mitochondria induced by acute or chronic intermittent hypoxia in the pre-Bötzinger complex of rats: an ultrastructural triple-labeling study with immunocytochemistry and histochemistry.

Introduction: The pre-Bötzinger complex (pre-BötC), a kernel of inspiratory rhythmogenesis, is a heterogeneous network with excitatory glutamatergic and inhibitory GABAergic and glycinergic neurons. Inspiratory rhythm generation relies on synchronous activation of glutamatergic neuron, whilst inhibitory neurons play a critical role in shaping the breathing pattern, endowing the rhythm with flexibility in adapting to environmental, metabolic, and behavioral needs. Here we report ultrastructural alterations in excitatory, asymmetric synapses (AS) and inhibitory, symmetric synapses (SS), especially perforated synapses with discontinuous postsynaptic densities (PSDs) in the pre-BötC in rats exposed to daily acute intermittent hypoxia (dAIH) or chronic (C) IH. Methods: We utilized for the first time a combination of somatostatin (SST) and neurokinin 1 receptor (NK1R) double immunocytochemistry with cytochrome oxidase histochemistry, to reveal synaptic characteristics and mitochondrial dynamic in the pre-BötC. Results: We found perforated synapses with synaptic vesicles accumulated in distinct pools in apposition to each discrete PSD segments. dAIH induced significant increases in the PSD size of macular AS, and the proportion of perforated synapses. AS were predominant in the dAIH group, whereas SS were in a high proportion in the CIH group. dAIH significantly increased SST and NK1R expressions, whereas CIH led to a decrease. Desmosome-like contacts (DLC) were characterized for the first time in the pre-BötC. They were distributed alongside of synapses, especially SS. Mitochondria appeared in more proximity to DLC than synapses, suggestive of a higher energy demand of the DLC. Findings of single spines with dual AS and SS innervation provide morphological evidence of excitation-inhibition interplay within a single spine in the pre-BötC. In particular, we characterized spine-shaft microdomains of concentrated synapses coupled with mitochondrial positioning that could serve as a structural basis for synchrony of spine-shaft communication. Mitochondria were found within spines and ultrastructural features of mitochondrial fusion and fission were depicted for the first time in the pre-BötC. Conclusion: We provide ultrastructural evidence of excitation-inhibition synapses in shafts and spines, and DLC in association with synapses that coincide with mitochondrial dynamic in their contribution to respiratory plasticity in the pre-BötC.

Ethanol-wet bonding technique may enhance the bonding performance of contemporary etch-and-rinse dental adhesives.

PURPOSE: To determine whether bonds of contemporary etch-and-rinse adhesives made with ethanol-wet bonding are stronger and more durable than those made with water-wet bonding, and to explore the possible reasons for the bonding results. MATERIALS AND METHODS: Flat surfaces of midcoronal dentin were made in extracted human third molars. The dentin surfaces were randomized into 6 groups according to bonding techniques (water- vs ethanol-wet bonding) and dental adhesives [Single Bond 2 (SB), Prime Bond NT (PB), and Gluma Comfort Bond (GB)]. After etching and rinsing, dentin surfaces were either left water-moist or immersed in ethanol. Following adhesive application and composite buildups, the bonded teeth were sectioned into beams for microtensile bond strength evaluation with or without NaOCl challenge. The morphology of the hybrid layer was analyzed with SEM. The wettability of water- vs. ethanol-saturated dentin was evaluated. The concentrations of non-volatile ingredients in the adhesives were compared. RESULTS: Compared to water-wet bonding, ethanol-wet bonding yielded similar (p > 0.05 for PB and GB) or higher (p < 0.05 for SB) 24-h bond strength, displayed significantly higher bond strength after chemical challenge (p < 0.05, for all three adhesives), and produced more even hybrid layers. Moreover, ethanol-saturated dentin exhibited a lower contact angle than water-saturated specimens, and the concentrations of non-volatile ingredients of the adhesives decreased in the order of SB > GB > PB. CONCLUSION: Ethanol-wet bonding could improve the bonding efficacy of contemporary etch-and-rinse adhesives, probably due to the good wettability of ethanol-saturated dentin and the structure of the hybrid layer. Moreover, this positive effect of ethanol-wet bonding might be influenced by the composition of adhesives.

Sevoflurane-Induced Neurotoxicity in the Developing Hippocampus via HIPK2/AKT/mTOR Signaling.

Sevoflurane (Sev) is a widely used inhalational anesthetic for general anesthesia in children. Previous studies have confirmed that multiple exposures to inhaled anesthetic can induce long-term neurotoxicity in newborn mice. However, the underlying mechanisms remain elusive. In this study, we investigated the role of homeodomain interacting protein kinase 2 (HIPK2), a stress activating kinase involved in neural survival and synaptic plasticity, and its underlying mechanism in sevoflurane-induced neurotoxicity. Empirical study showed that neuronal apoptosis was elevated after exposure to sevoflurane. Meanwhile, up-regulation of HIPK2 and AKT/mTOR signaling was observed in primary hippocampal neurons and hippocampus in mice upon anesthetic exposure. A64, antagonist of HIPK2, could significantly reduce increased apoptosis and activation of AKT/mTOR induced by sevoflurane. AKT antagonist MK2206 partially alleviated neuronal apoptosis without affecting the expression of HIPK2. Experimental results demonstrated a crucial role of HIPK2/AKT/mTOR signaling in neurotoxicity of sevoflurane. Thus, HIPK2/AKT/mTOR signaling can serve as a potential target for the protection of inhalation anesthesia-induced cytotoxicity in the future.

Bomidin: An Optimized Antimicrobial Peptide With Broad Antiviral Activity Against Enveloped Viruses.

The rapid evolution of highly infectious pathogens is a major threat to global public health. In the front line of defense against bacteria, fungi, and viruses, antimicrobial peptides (AMPs) are naturally produced by all living organisms and offer new possibilities for next-generation antibiotic development. However, the low yields and difficulties in the extraction and purification of AMPs have hindered their industry and scientific research applications. To overcome these barriers, we enabled high expression of bomidin, a commercial recombinant AMP based upon bovine myeloid antimicrobial peptide-27. This novel AMP, which can be expressed in Escherichia coli by adding methionine to the bomidin sequence, can be produced in bulk and is more biologically active than chemically synthesized AMPs. We verified the function of bomidin against a variety of bacteria and enveloped viruses, including severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), herpes simplex virus (HSV), dengue virus (DENV), and chikungunya virus (CHIKV). Furthermore, based on the molecular modeling of bomidin and membrane lipids, we elucidated the possible mechanism by which bomidin disrupts bacterial and viral membranes. Thus, we obtained a novel AMP with an optimized, efficient heterologous expression system for potential therapeutic application against a wide range of life-threatening pathogens.

Autophagic LC3+ calcified extracellular vesicles initiate cartilage calcification in osteoarthritis.

Pathological cartilage calcification plays an important role in osteoarthritis progression but in which the origin of calcified extracellular vesicles (EVs) and their effects remain unknown. Here, we demonstrate that pathological cartilage calcification occurs in the early stage of the osteoarthritis in which the calcified EVs are closely involved. Autophagosomes carrying the minerals are released in EVs, and calcification is induced by those autophagy-regulated calcified EVs. Autophagy-derived microtubule-associated proteins 1A/1B light chain 3B (LC3)-positive EVs are the major population of calcified EVs that initiate pathological calcification. Release of LC3-positive calcified EVs is caused by blockage of the autophagy flux resulted from histone deacetylase 6 (HDAC6)-mediated microtubule destabilization. Inhibition of HDAC6 activity blocks the release of the LC3-positive calcified EVs by chondrocytes and effectively reverses the pathological calcification and degradation of cartilage. The present work discovers that calcified EVs derived from autophagosomes initiate pathological cartilage calcification in osteoarthritis, with potential therapeutic targeting implication.

Mitochondria transplantation protects traumatic brain injury via promoting neuronal survival and astrocytic BDNF.

Traumatic brain injury (TBI) is one of the leading causes of disability and paralysis around the world. Secondary injury, characterized by progressive neuronal loss and astrogliosis, plays important roles in the post-TBI cognitive impairment and mood disorder. Unfortunately, there still lacks effective treatments, particularly surgery interferences for it. Recent findings of intercellular mitochondria transfer implies a potential therapeutic value of mitochondria transplantation for TBI, which has not been tested yet. In the present study, we demonstrated a quick dysfunction of mitochondria, up-regulation of Tom20 in the injured cortex and subsequent cognitive and mood impairment. Our data demonstrated that mitochondria derived from allogeneic liver or autogeneic muscle stimulated similar microglial activation in brain parenchyma. In vitro experiments showed that exogenous mitochondria could be easily internalized by neurons, astrocytes, and microglia, except for oligodendrocytes. Mitochondria transplantation effectively rescued neuronal apoptosis, restored the expression of Tom20 and the phosphorylation of JNK. Further analysis revealed that mitochondria transplantation in injured cortex induced a significant up-regulation of BDNF in reactive astrocytes, improved animals' spatial memory and alleviated anxiety. In together, our data indicate that mitochondria transplantation may has the potential of clinical translation for TBI treatment, in combination with surgery.

Endocannabinoid system in the neurodevelopment of GABAergic interneurons: implications for neurological and psychiatric disorders.

In mature mammalian brains, the endocannabinoid system (ECS) plays an important role in the regulation of synaptic plasticity and the functioning of neural networks. Besides, the ECS also contributes to the neurodevelopment of the central nervous system. Due to the increase in the medical and recreational use of cannabis, it is inevitable and essential to elaborate the roles of the ECS on neurodevelopment. GABAergic interneurons represent a group of inhibitory neurons that are vital in controlling neural network activity. However, the role of the ECS in the neurodevelopment of GABAergic interneurons remains to be fully elucidated. In this review, we provide a brief introduction of the ECS and interneuron diversity. We focus on the process of interneuron development and the role of ECS in the modulation of interneuron development, from the expansion of the neural stem/progenitor cells to the migration, specification and maturation of interneurons. We further discuss the potential implications of the ECS and interneurons in the pathogenesis of neurological and psychiatric disorders, including epilepsy, schizophrenia, major depressive disorder and autism spectrum disorder.

Brain-Type Glycogen Phosphorylase Is Crucial for Astrocytic Glycogen Accumulation in Chronic Social Defeat Stress-Induced Depression in Mice.

Astrocytic glycogen plays an important role in brain energy metabolism. However, the contribution of glycogen metabolism to stress-induced depression remains unclear. Chronic social defeat stress was used to induce depression-like behaviors in mice, assessed with behavioral tests. Glycogen concentration in the medial prefrontal cortex (mPFC) and the expression of key enzymes of the glycogen metabolism were investigated using Western blots, immunofluorescent staining, electron microscopy, and biochemical assays. Stereotaxic surgery and viral-mediated gene transfer were applied to knockdown or overexpress brain-type glycogen phosphorylase (PYGB) in the mPFC. The glycogen content increased in the mPFC after stress. Glycogenolytic dysfunction due to inactivation of PYGB was responsible for glycogen accumulation. Behavioral tests on astrocyte-specific PYGB overexpression mice showed that augmenting astrocytic PYGB reduces susceptibility to depression when compared with stress-susceptible mice. Conversely, PYGB genetic down-regulation in the mPFC was sufficient to induce glycogen accumulation and depression-like behaviors. Furthermore, PYGB overexpression in the mPFC decreases susceptibility to depression, at least partially by rescuing glycogen phosphorylase activity to maintain glycogen metabolism homeostasis during stress. These findings indicate that (1) glycogen accumulation occurs in mice following stress and (2) glycogenolysis reprogramming leads to glycogen accumulation in astrocytes and PYGB contributes to stress-induced depression-like behaviors. Pharmacological tools acting on glycogenolysis might constitute a promising therapy for depression.

Necroptotic astrocytes induced neuronal apoptosis partially through EVs-derived pro-BDNF.

Our previous study showed that neuronal apoptosis was significantly increased upon treatment of conditioned medium (CM) from necroptotic astrocytes (NAS), leaving the underlying mechanism unclear. Considering the nutritive and supportive roles of astrocytes, we first examined the neurotrophic phenotype of necroptotic astrocytes with focus on glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF), two important neurotrophic factors, and it was unexpectedly found that the expression of GDNF and BDNF were up-regulated in necroptotic astrocytes in vitro. A question was raised as to whether the functional secreted forms of neurotrophic factors were increased. Considering that extracellular vesicles (EVs) were carriers of secreted substances and their roles in cellular interaction, we isolated EVs from astrocytes and found EVs from normal and necroptotic astrocytes (EVs-NAS) had characteristics of exosomes. We then examined GDNF and BDNF in EVs-NAS, and BDNF was interestingly found as an immature form of pro-BDNF. The expression of pro-BDNF was found to be increased in EVs-NAS, and EVs-NAS had a negative effect on neuronal survival. To verify that whether pro-BDNF was involved in the detrimental effect of EVs-NAS, anti-pro-BDNF antibody was applied, and we found that neuronal apoptosis-induced by EVs-NAS could be significantly attenuated by blocking pro-BDNF, which suggested that necroptotic astrocytes induced neuronal apoptosis partially through EVs-derived pro-BDNF. The data expand our understanding in neurotrophic phenotype of necroptotic astrocytes, and may provide us new strategies targeting on EVs-NAS in treatment of neurological diseases.

HTNV infection of CD8+ T cells is associated with disease progression in HFRS patients.

Hantaan viruses (HTNVs) are zoonotic pathogens transmitted mainly by rodents and capable of infecting humans. Increasing knowledge of the human response to HTNV infection can guide the development of new preventative vaccines and therapeutic strategies. Here, we show that HTNV can infect CD8+ T cells in vivo in patients diagnosed with hemorrhagic fever with renal syndrome (HFRS). Electron microscopy-mediated tracking of the life cycle and ultrastructure of HTNV-infected CD8+ T cells in vitro showed an association between notable increases in cytoplasmic multivesicular bodies and virus production. Notably, based on a clinical cohort of 280 patients, we found that circulating HTNV-infected CD8+ T cell numbers in blood were proportional to disease severity. These results demonstrate that viral infected CD8+ T cells may be used as an adjunct marker for monitoring HFRS disease progression and that modulating T cell functions may be explored for new treatment strategies.