Journal of Biomedical Science最新文献

Extracellular vesicles (EVs) are heterogeneous populations of membrane-bound particles released from almost all cell types in an organism and play pivotal roles in cell-cell communication. EVs carry nucleic acids, proteins, metabolites and other bioactive substances, which are taken by the recipient cells to alter cell physiology and functions. The cargo landscapes of EVs are influenced by the cell contexts and the biogenesis mechanisms of EVs, in which certain molecules govern both biogenesis and cargo sorting. In this review, we discuss the biogenesis and secretion mechanisms of various types of EVs, including several atypical EVs. In addition, given that the tumor immune microenvironment (TIME) is intricately controlled by the communication between tumor cells and various immune cells, we summarize the latest update about how tumor-derived EVs influence the phenotypes of various immune cells in tumor microenvironment for tumor immune evasion, and, conversely, how EVs secreted from immune cells in TIME control the malignancies of tumor cells. In particular, we discuss the roles of several atypical EVs in regulating TIME. Lastly, we highlight the advantages of utilizing EVs as liquid biopsies for cancer diagnosis, the application and challenge of EVs in different anti-tumor therapies, and the recent clinical trials that exploit EVs as drug carriers. As the continuous advances in our understanding of the complex biogenesis mechanisms and the pleiotropic actions of EVs in TIME as well as the technology improvements in harnessing EVs' clinical benefits, we can expect to further unlock the biomedical potential of EVs in cancer and other diseases.

Background: Brain-derived neurotrophic factor (BDNF) is a key mediator of synaptic plasticity and memory formation in the hippocampus. However, the BDNF-induced alterations in the glutamate receptors coupled to the plasticity of glutamatergic synapses in the hippocampus have not been elucidated. In this work we investigated the putative role of GluN2B-containing NMDA receptors in the plasticity of glutamatergic synapses induced by BDNF.

Methods: The effects of BDNF on the surface expression of GluN2B-containing NMDA receptors was investigated in cultured hippocampal neurons and in hippocampal synaptoneurosomes by immunocytochemistry under non-permeabilizing conditions, using an antibody that binds to an extracellular epitope. Long term potentiation of hippocampal CA1 synapses was induced by using θ-burst stimulation. Epileptic seizures were induced using the Li⁺-pilocarpine model of temporal lobe epilepsy. Pyk2 phosphorylation was assessed by western blot with a phosphospecific antibody.

Results: Stimulation of hippocampal synaptoneurosomes with BDNF led to a significant time-dependent increase in the synaptic surface expression of GluN2B-containing NMDA receptors as determined by immunocytochemistry with colocalization with pre- (vesicular glutamate transporter) and post-synaptic markers (PSD-95). Similarly, BDNF induced the synaptic accumulation of GluN2B-containing NMDA receptors at the synapse in cultured hippocampal neurons, by a mechanism sensitive to the PKC inhibitor GӦ6983. The effects of PKC may be mediated by phosphorylation of Pyk2, as suggested by western blot experiments analyzing the phosphorylation of the kinase on Tyrosine 402. GluN2B-containing NMDA receptors mediated the effects of BDNF in the facilitation of the early phase of long-term potentiation (LTP) of hippocampal CA1 synapses induced by θ-burst stimulation, since the effect of the neurotrophin was abrogated in the presence of the GluN2B inhibitor Co 101244. In the absence of BDNF, the GluN2B inhibitor did not affect LTP. Surface accumulation of GluN2B-containing NMDA receptors was also observed in hippocampal synaptoneurosomes isolated from rats subjected to the pilocarpine model of temporal lobe epilepsy, after reaching Status Epilepticus, an effect that was inhibited by administration of the TrkB receptor inhibitor ANA-12.

Conclusion: Together, these results show that the synaptic accumulation of GluN2B-containing NMDA receptors mediate the effects of BDNF in the plasticity of glutamatergic synapses in the hippocampus.

Aging is the foremost risk factor for metabolic syndrome and atherosclerosis, which is a principal cause of cardiovascular diseases (CVDs). Vascular endothelial cells (ECs), which line the vascular intima, play a central role in maintaining vascular homeostasis. Their dysfunction, marked by impaired barrier function, inflammation, and metabolic dysregulation, constitutes an early and pivotal event in atherogenesis. As key sensors of hemodynamic forces, ECs are constantly exposed to blood flow-induced shear stress, which exert divergent effects on metabolism depending on the flow pattern. Laminar flow with relatively high shear stress (LS), as a critical atheroprotective factor, maintains EC quiescence and promotes anti-inflammatory responses and antioxidant defense, whereas disturbed flow with low and oscillatory shear stress (OS), induces the athero-susceptible signaling network to activate glycolysis and inflammation in ECs. While genetic, epigenetic, and molecular signaling mechanisms in EC physiology and pathophysiology have been extensively explored, the crucial role of EC metabolism in EC dysfunction and atherogenesis remains largely understudied. By serving as precursors, intermediates, and end products of cellular processes, metabolites offer a dynamic snapshot of endothelial metabolic states under both physiological and pathophysiological conditions. With aging, ECs undergo profound metabolic reprogramming, including disrupted glycolysis, mitochondrial dysfunction, and altered redox homeostasis. In healthy vasculature, ECs maintain quiescence and metabolic homeostasis, primarily relying on glycolysis for energy. With aging, the gradual accumulation of atherosclerotic risk factors, including oxidative stress, inflammation, dyslipidemia, and hyperglycemia, drives metabolic reprogramming in ECs, particularly in regions exposed to disturbed flow with OS, ultimately leading to EC dysfunction and atherosclerosis. This review summarizes recent advances in age-related metabolic reprogramming in ECs and its contribution to atherosclerosis, particularly focusing on the dysregulation of glycolysis, fatty acid metabolism, amino acid metabolism, and mitochondrial respiration induced by age and fluid shear stress. This review also outlines recent methodologies for profiling EC metabolism, and discusses potential therapeutic applications of targeting EC metabolism to prevent or delay the development of atherosclerosis.

Background: Growing evidence highlights the importance of epitranscriptomic regulation in cerebellar development and function, especially through m⁶A methylation. Nevertheless, the precise function of the RNA demethylase Fto in the cerebellum is still uncertain.

Methods: An Fto knockout (Fto^KO) mouse model was generated to investigate the role of Fto in cerebellar development. Cerebellar function was assessed using the behavioral tests and Nissl staining. Immunofluorescence was performed to detect molecular expression levels and subcellular localization. Dot blot, m⁶A-RIP-seq, ATAC-seq and CUT&Tag-seq were used to confirm m⁶A levels and chromatin accessibility. Co-IP was employed to test molecular interactions.

Results: Fto^KO mice exhibited cerebellar ataxia, including tremors and abnormal gait patterns. Reduced FTO expression at embryonic day 13.5 (E13.5) and postnatal day 3 (P3) stages resulted in increased TUJ1 expression, as well as reductions in neuronal functional genes (Map2) and self-renewal genes (Sox2, Sox9, Nestin and Pax6). Mechanistically, Kat8 upregulation was linked to the high m⁶A levels regulated by Fto loss. Furthermore, IGF2BP3 specifically recruited acetyltransferase KAT8 to control gene transcription during early cerebellar development by regulating H4K16ac modification, which alters chromatin accessibility in neural developmental pathways.

Conclusions: In summary, Fto^KO-induced Kat8 upregulation in an m⁶A-dependent manner resulted in enhanced KAT8 recruitment by IGF2BP3, which improved chromatin accessibility and H4K16ac modification, thereby promoting cerebellar developmental dysfunction.

Background: Triple-negative breast cancer (TNBC) has the highest mortality rate among all breast cancer subtypes. Although immunotherapy shows promise, its efficacy varies. CDK4/6 inhibitors can radiosensitize and modulate the immune system, and high-dose radiotherapy (RT) can enhance the effects of immunotherapy. This study explored the combination of RT with CDK4/6 inhibitors to improve TNBC immunotherapy by modulating the tumor microenvironment.

Methods: We assessed the radiosensitizing effects of abemaciclib (a CDK4/6 inhibitor) using clonogenic assays in three human TNBC cell lines (MDA-MB-231, MDA-MB-453, and MDA-MB-468) and two murine TNBC cell lines (4T1 and EMT6). The antitumor efficacy of the treatments (control, RT, abemaciclib, anti-PD-L1 antibody [aPD-L1], abemaciclib combined with aPD-L1, abemaciclib combined with RT, aPD-L1 combined with RT, and the triple combination of abemaciclib with aPD-L1 and RT) was evaluated in both 4T1 and EMT6 cell line-derived immunocompetent mouse models. Interferon-γ (IFN-γ) levels in mouse blood were monitored to gauge the immune response. Tumor-infiltrating lymphocytes (TILs) were analyzed using flow cytometry and immunohistochemical staining.

Results: Clonogenic assays showed synergistic effects of RT and abemaciclib in all TNBC cell lines. RT increased PD-L1 expression, whereas abemaciclib did not alter PD-L1 expression. In the 4T1 and EMT6 mouse models, the triple combination treatment markedly inhibited tumor growth (P < 0.01). In the 4T1 mouse model, the triple combination group exhibited significantly greater circulating IFN-γ levels (P < 0.001) than the other groups. TIL analysis revealed a significant increase in CD4 + and CD8 + T cells and tumor-associated macrophages (P < 0.01) in the triple combination therapy group. Immunohistochemical staining confirmed increased infiltration of CD4 + T cells, CD8 + T cells, monocyte chemoattractant protein-1, CD80-, and iNOS- positive macrophages into the tumor microenvironment of this group, with a marked reduction in CD206-positive macrophages.

Conclusion: Combining CDK4/6 inhibitors with RT enhanced the antitumor effects of aPD-L1 immunotherapy against TNBC. This effect was correlated with increased IFN-γ secretion and recruitment of CD4 + and CD8 + T cells and M1 tumor-associated macrophages, leading to modulation of the tumor microenvironment.

Background: The pineal gland produces melatonin to control circadian rhythm via the final enzyme in the serotonin pathway, hydroxyindole O-methyltransferase (HIOMT). Interestingly, HIOMT is expressed by certain non-pineal cells. The main catalytically active of the three human HIOMT (hHIOMT) isoforms in pineal cells is hHIOMT345 (345 amino acids), while hHIOMT298 (298 amino acids) is the most active isoform in fibroblasts, where it converts 5-hydroxytryptophan to 5-methoxytryptophan (5-MTP). We previously demonstrated that exogenous 5-MTP protects the arteries. Nevertheless, whether vascular smooth muscle cells (VSMCs) per se synthesize 5-MTP is unknown.

Methods: We transfected primary wild-type VSMCs with different hHIOMT isoforms and treated them with inflammatory cytokines to examine hHIOMT's effects on p38 MAPK activation. Global and VSMC-specific hHIOMT transgenic mice were generated and subjected to an arterial injury model. Histological analysis was performed to evaluate intimal hyperplasia and expression of select tryptophan metabolites and their synthetic enzymes. We treated wild-type and transgenic VSMCs with interleukin-1 beta (IL-1β), with or without 5-MTP, to examine the levels of serotonin and aromatic L-amino acid decarboxylase (AADC). Serotonin's effects on VSMC functions were evaluated, and inhibitors of p38 MAPK and ERK1/2 were used to determine the signaling pathways. The effects of AADC on VSMCs were assessed by AADC knockdown or overexpression.

Results: Overexpression of the human full-length isoform of 373 amino acids (hHIOMT373) in VSMCs attenuated proinflammatory cytokine-induced p38 MAPK activation, similar to 5-MTP treatment. Global and VSMC-specific hHIOMT373 transgenic mice exhibited attenuated intimal hyperplasia following arterial injury. Intriguingly, the tryptophan metabolite serotonin and its synthetic enzyme AADC were reduced in transgenic arteries. In VSMCs, IL-1β increased AADC and serotonin levels that were mitigated by 5-MTP treatment or HIOMT overexpression via suppressing the p38 MAPK pathway. Interestingly, serotonin promoted VSMC proliferation and decreased VSMC marker levels through ERK1/2 activation. While AADC overexpression decreased VSMC contractile markers, AADC knockdown suppressed IL-1β-induced VSMC proliferation.

Conclusions: Our results unveiled a unique function of HIOMT in vascular disease. In VSMCs, hHIOMT373 reprogrammed tryptophan metabolism to increase 5-MTP and decrease serotonin levels, thereby protecting against injury-induced intimal hyperplasia. Mechanistically, HIOMT-5-MTP suppressed AADC-serotonin induction through inhibiting p38 MAPK activation.

Background: Postnatal skeletal muscle development leads to increased muscle mass, strength, and mitochondrial function, but the role of mitochondrial remodeling during this period is unclear. This study investigates mitochondrial remodeling during postnatal muscle development and examines how constitutive autophagy deficiency impacts these processes.

Methods: We initially performed a broad RNA-Seq analysis using a publicly available GEO database of skeletal muscle from postnatal day 7 (P7) to postnatal day 112 (P112) to identify differentially expressed genes. This was followed by investigation of postnatal skeletal muscle development using the mitophagy report mouse line (mt-Kiema mice), as well as conditional skeletal muscle knockout (Atg7^{f/f:Acta1-Cre}) mice.

Results: Our study observed rapid growth of body and skeletal muscle mass, along with increased fiber cross-sectional area and grip strength. Mitochondrial maturation was indicated by enhanced maximal respiration, reduced electron leak, and elevated mitophagic flux, as well as increased mitochondrial localization of autophagy and mitophagy proteins. Anabolic signaling was also upregulated, coinciding with increased mitophagy and fusion signaling, and decreased biogenesis signaling. Despite the loss of mitophagic flux in skeletal muscle-specific Atg7 knockout mice, there were no changes in body or skeletal muscle mass; however, hypertrophy was observed in type IIX fibers. This lack of Atg7 and loss of mitophagy was associated with the activation of mitochondrial apoptotic signaling as well as ubiquitin-proteasome signaling, suggesting a shift in degradation mechanisms. Inhibition of the ubiquitin-proteasome system (UPS) in autophagy-deficient skeletal muscle led to significant atrophy, increased reactive oxygen species production, and mitochondrial apoptotic signaling.

Conclusion: These results highlight the role of mitophagy in postnatal skeletal muscle development and suggest that autophagy-deficiency triggers compensatory degradative pathways (i.e., UPS) to prevent mitochondrial apoptotic signaling and thus preserve skeletal muscle integrity in developing mice.

Background: Rickettsiosis is among the deadliest vector-borne infectious diseases worldwide, in part because rickettsiae replicate within human cells, where antibodies and most drugs cannot effectively reach this obligatory intracellular pathogen. Ehrlichia chaffeensis, an emerging rickettsia, is the causative agent of human monocytic ehrlichiosis. We therefore aim to generate intrabodies (IBs), the variable domain of heavy chain of heavy-chain-only antibodies (VHHs) that bind intracellular bacterial proteins to inhibit E. chaffeensis infection.

Methods: E. chaffeensis replicates in membrane-bound vacuoles resembling early endosomes in human monocytes/macrophages. The type IV secretion system effector Ehrlichia translocated factor-2 (Etf-2) directly binds to RAB5-GTP on E. chaffeensis-containing vacuoles. Consequently, Etf-2 hinders the engagement of RAB5 GTPase-activating protein with RAB5-GTP, delays maturation of Ehrlichia vacuoles to late endosomes, thus facilitates infection. As C-terminal half of Etf-2 (Etf-2C) binds RAB5-GTP, a random synthetic library of VHHs was screened for binding to Etf-2C, and for inhibition of Etf-2 binding to RAB5 in human cells when expressed intracellularly (IBs). Positive IBs were tested for inhibition of Etf-2 functions and E. chaffeensis infection, and lipid nanoparticles-encapsulated mRNAs (mRNAs-LNP) platform was used to deliver IBs in vitro and in mice.

Results: We have identified two distinct IBs that inhibit Etf-2 binding to RAB5 and Etf-2 functions in vitro. Synthesized mRNA-LNP encoding anti-Etf-2 IBs significantly inhibited E. chaffeensis infection in cell cultures and in a mouse model.

Conclusions: The results demonstrate the feasibility of mRNA-LNP encoding IBs as intracellular probes and a precision therapy addressing underlying cause of obligatory intracellular infection.

Background: Therapeutic options against metallo-β-lactamase producing P. aeruginosa (MBL-PA) are limited due to multi-drug resistance. A jumbo phage isolated from wastewater in Greece was characterized microbiologically and genetically and evaluated for its potential as a therapeutic agent alone or in combination with antibiotics in an experimental thigh infection mouse model.

Methods: The host range of the jumbo phage vB_PaerM_AttikonH10 (AttikonH10) against 20 MBL-PA clinical isolates and 10 susceptible strains, one-step phage growth and growth curves of mid-exponential phase bacteria upon addition of the phage were analyzed. Whole-genome sequencing was performed and the de novo assembled complete phage genome was compared with other jumbo phages. In vivo pharmacokinetics in different tissues as well as the efficacy of two dosing regimens 10⁹ and 10⁶ PFU/mouse administered intraperitoneally alone and in combination with amikacin (384 mg/kg/day) was tested against an MBL-PA clinical isolate in murine thigh infection model.

Results: The phage formed small plaques in double-layer agar and demonstrated clear or semi-clear lysis in 83.3% (25/30) of P. aeruginosa clinical isolates. Growth curves showed a > 94% growth inhibition in the presence of phage even at the lowest multiplicity of infection ratio tested (10^-5). Whole genome analysis indicated a jumbo dsDNA phage with 278,406 bp (36.92% GC) belonging to Phikzvirus that is predicted to host up to 413 putative ORFs and 6 tRNA genes. No known lysogeny-associated genes, virulence factors, or antimicrobial resistance genes were identified within the genome. Phage titres 10⁴-10⁶ PFU/tissue were detected in all mouse tissues with elimination half-life of 3.4 h except in bronchoalveolar lavage where no phages were found. Only the high phage dose (10⁹ PFU/mouse) reduced bacterial load in thigh by 1.09 log₁₀ cfu/thigh compared to placebo, similar to amikacin monotherapy (1.19 log₁₀ cfu/thigh), while their combination achieved a greater reduction of 2.07 log₁₀ cfu/thigh compared to each monotherapy (p = 0.0044-0.0048).

Conclusions: The newly reported Phikzvirus jumbo phage AttikonH10 demonstrated a broad host range, strong lytic activity and synergistic effects with amikacin against MBL-PA isolates making it a candidate for phage therapy.