Journal of Biomedical Science最新文献

Background: The evolution of SARS-CoV-2, particularly through new variants, presents significant global health challenges due to their potential for immune evasion and reduced vaccine effectiveness. This study aims to investigate the impact of mutations in the Spike protein of Omicron EG.5 and XBB.1.16 variants on the binding affinities of viral peptides to common human leukocyte antigen (HLA) class I and II alleles across Taiwanese, British, and Russian populations. Understanding these interactions is crucial for elucidating differences in immune responses and disease severity among diverse populations.

Methods: We updated the T-CoV portal to incorporate and analyze EG.5 and XBB.1.16 variants. Binding affinities between mutated Spike protein peptides and HLA class I and II alleles were predicted and compared across the three populations. Statistical analyses, including chi-squared tests, were conducted to assess the significance of binding affinity differences across the three populations and between HLA classes.

Results: Our findings revealed that mutations in the Spike protein had a more pronounced effect on HLA class II binding affinities than on HLA class I. While binding affinity profiles for HLA class I were largely consistent across populations, significant population-specific variations were observed for HLA class II alleles. Specifically, the British population exhibited lower proportions of tightly binding mutated peptides compared to the Taiwanese and Russian populations. Furthermore, substantial differences were identified in the binding affinity changes of mutated Spike peptides for HLA class II across Taiwanese, British, and Russian populations, as well as between the Omicron EG.5 and XBB.1.16 variants. Subsequent analyses revealed significant differences in the conservation and evolutionary trajectories of binding affinities between mutated Spike peptides and common HLA class II alleles, both between the EG.5 and XBB.1.16 variants and across the three populations for the XBB.1.16 variant.

Conclusions: In summary, Spike protein mutations in SARS-CoV-2 variants significantly influence immune responses by altering HLA-peptide interactions, with pronounced population-specific effects on HLA class II alleles. These findings underscore the critical role of HLA class II diversity in shaping immune responses and susceptibility to COVID-19. Integrating population-specific HLA profiles into vaccine development and public health strategies is essential for improving interventions against evolving SARS-CoV-2 variants.

Background: Anxiety is an innate response in the face of danger. When anxiety is overwhelming or persistent, it could be considered an anxiety disorder. Recent studies have shown that acid-sensing ion channels (ASICs) represent a novel class of promising targets for developing effective therapies for anxiety. Especially, ASIC1a and ASIC4 of the ASIC family are widely expressed in the central nervous system and their gene knockouts result in reducing or enhancing anxiety-like responses in mice respectively. However, how ASIC1a and ASIC4 modulate anxiety-associated responses remains unknown.

Methods: Here we combined chemo-optogenetic, conditional knockout, gene rescue, molecular biology and biochemistry, and electrophysiological approaches to probe the roles of ASIC4 and ASIC4-expressing cells in anxiety-associated responses in mouse models.

Results: Chemo-optogenetically activating ASIC4-positive cells induced fear and anxiety-like responses in mice. Also, mice lacking ASIC4 (Asic4^-/-) in the amygdala or the bed nucleus of the stria terminalis (BNST) exhibited anxiety-associated phenotypes. Conditional knockout of ASIC1a in ASIC4-positive cells reduced anxiety-associated behaviors. In situ hybridization analyses indicated that ASIC4 transcripts were highly co-localized with ASIC1a in the amygdala and BNST. We identified two glycosylation sites of ASIC4, Asn191 and Asn341, that were involved in interacting with ASIC1a and thus could modulate ASIC1a surface protein expression and channel activity. More importantly, viral vector-mediated gene transfer of wild-type ASIC4 but not Asn191 and Asn341 mutants in the amygdala or BNST rescued the anxiogenic phenotypes of Asic4^-/- mice.

Conclusions: Together, these data suggest that ASIC4 plays an important role in fear and anxiety-related behaviors in mice by modulating ASIC1a activity in the amygdala and BNST.

Metabolic reprogramming enables tumour cells to sustain their continuous proliferation and adapt to the ever-changing microenvironment. Branched-chain amino acids (BCAAs) and their metabolites are involved in intracellular protein synthesis and catabolism, signal transduction, epigenetic modifications, and the maintenance of oxidative homeostasis. Alterations in BCAA metabolism can influence the progression of various tumours. However, how BCAA metabolism is dysregulated differs among depending on tumour type; for example, it can manifest as decreased BCAA metabolism leading to BCAA accumulation, or as enhanced BCAA uptake and increased catabolism. In this review, we describe the role of BCAA metabolism in the progression of different tumours. As well as discuss how BCAA metabolic reprogramming drives tumour therapy resistance and evasion of the antitumour immune response, and how these pro-cancer effects are achieved in part by activating the mTORC signalling pathway. In-depth investigations into the potential mechanisms by which BCAA metabolic reprogramming affects tumorigenesis and tumour progression can enhance our understanding of the relationship between metabolism and cancer and provide new strategies for cancer therapy.

Background: Mutations in the HERG potassium channel are a major cause of long QT syndrome type 2 (LQT2), which can lead to sudden cardiac death. The HERG channel plays a critical role in the repolarization of the myocardial action potential, and loss-of-function mutations prolong cardiac repolarization.

Methods: In this study, we investigated the efficacy and underlying molecular mechanism of ICA-105574, an HERG activator, in shortening the duration of cardiac repolarization in severe LQT2 variants. We characterized the efficacy of ICA-105574 in vivo, using an animal model to assess its ability to shorten the QT interval and in vitro, in cellular models mimicking severe HERG channel mutations (A561V, G628S, and L779P) to evaluate its impact in enhancing I_Kr current. Additionally, molecular dynamics simulations were used to investigate the molecular mechanism of ICA-105574 action.

Results: In vivo, ICA-105574 significantly shortened the QT interval. LQT2 mutations drastically reduced I_Kr amplitude and suppressed tail currents in cellular models. ICA-105574 restored I_Kr in A561V and G628S. Finally, in silico data showed that ICA-105574 stabilizes a pattern of interactions similar to gain-of-function SQT1 mutations and can reverse the G628S modifications, through an allosteric network linking the binding site to the selectivity filter and the S5P turret helix, thereby restoring its K⁺ ion permeability.

Conclusions: Our results support the development of HERG activators like ICA-105574 as promising pharmacological molecules against some severe LQT2 mutations and suggest that molecular dynamics simulations can be used to test the ability of molecules to modulate HERG function in silico, paving the way for the rational design of new HERG activators.

Background: Phosphorylation and O-GlcNAcylation are the key modifications regulating RNA Polymerase II (RNA Pol II)-driven transcription. Transcriptional kinases, cyclin-dependent kinase 7 (CDK7), CDK9 and CDK12 phosphorylate RNA Pol II, whereas O-GlcNAcylation is added by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA). Currently, no study has systematically evaluated how inhibiting each of these enzyme activities impacts the assembly of the appropriate protein complexes on the polymerase and the maturation of mRNA.

Methods: Here, we systematically evaluate remodeling of RNA Pol II interactome and effects on the nascent mRNA maturation by using mass spectrometry and SLAM-seq, respectively. For validation, we rely predominantly on analysis of intronic polyadenylation (IPA) sites, mitochondrial flux assays (Seahorse), western blotting and patient data.

Results: We show that OGT / OGA inhibition reciprocally affect protein recruitment to RNA Pol II, and appropriate O-GlcNAcylation levels are required for optimal function of the RNA Pol II complex. These paradoxical effects are explained through IPA, because despite being prematurely poly-adenylated, these mRNAs are scored as mature in SLAM-seq. Unlike previously proposed, we show that, similar to inhibition of CDK12, also targeting CDK9 stimulates transcription of short genes at the cost of long genes. However, our systematic proteomic- and IPA-analysis revealed that these effects are mediated by distinct molecular mechanisms: CDK9 inhibition leads to a failure of recruiting Integrator complex to RNA Pol II, and we then show that depletion of Integrator subunits phenocopy the gene length-dependent effects. In contrast, CDK12 inhibition triggers IPA. Finally, we show that dynamic O-GlcNAcylation predominantly interplays with CDK9: OGT inhibition augments CDK9 inhibitor effects on mRNA maturation due to defects in transcription elongation, while OGA inhibition rescues mRNA maturation failure caused by targeting CDK9, but induces IPA.

Conclusion: We show that dynamic O-GlcNAcylation is a negative regulator of mRNA biosynthesis and propose that the addition and removal of the modification serve as quality control-steps to ascertain successful generation of mature mRNAs. Our work identifies unprecedented redundancy in the regulation of RNA Pol II, which increases resilience towards transcriptional stress, and also underscores the difficulty of targeting transcription to control cancer.

Backgrounds: Endolysins are promising alternatives to antibiotics because they can lyse bacterial cells rapidly with a low risk of resistance development, however, their effectiveness against Gram-negative bacteria is hindered by the presence of the outer membrane present in Gram-negative bacteria. Several endolysins with amphipathic helices at the C-terminus have been reported to have intrinsic antibacterial activity against Gram-negative bacteria but their action mechanism is not fully elucidated.

Methods: The sequence alignment analysis was assessed with the CLC Main workbench 7, and His-tagged endolysins were purified with affinity chromatography. Site-directed mutagenesis was used to generate mutations in the endolysin to make various endolysin mutants. The muralytic activity of the endolysin against Gram-negative bacteria was analyzed using a turbidity reduction assay and the antibacterial activities of the endolysins were assessed through a viable cell counting assay.

Results: We identified two endolysins, LysTS3 and LysTS6, both of which have similar sequences and structures including the amphipathic helices at their C-terminus. LysTS6 exhibited significantly higher antibacterial activity against Gram-negative bacteria compared to LysTS3 even though both enzymes have similar muralytic activity against the outer membrane-permeabilized Gram-negative bacteria. Systematic truncation and bioinformatic analysis of these two endolysins revealed a major difference in the charge on the surface of their C-terminal helices, suggesting the possibility that the charge on this helix can determine the antibacterial activity of the endolysins against Gram-negative bacteria. We could enhance the activity of LysTS3 against Gram-negative bacteria by replacing Ala₁₅₆ and Glu₁₆₀ with lysine and alanine, respectively, the amino acid residues at the structurally equivalent positions in LysTS6. A similar activity boost was also seen in LysSPN1S and LysJEP4 when the surface charge of the C-terminal amphipathic helix was altered to be more positive through the modification of the surface-exposed amino acid residues.

Conclusions: The antibacterial activity of endolysin against Gram-negative bacteria could be enhanced by adjusting the surface charge on the C-terminal amphipathic helix to more positive, suggesting that the positive surface charge on the C-terminal amphipathic helix of endolysin is crucial for its penetration of outer membrane to reach peptidoglycan layer of Gram-negative bacteria.

Background: Triple-negative breast cancer (TNBC) is characterized by high malignancy, strong invasiveness, and a propensity for distant metastasis, leading to poor prognosis and relatively limited treatment options. Metformin, as a first-line oral hypoglycemic agent, has garnered widespread research interest in recent years due to its potential in cancer prevention and treatment. However, its efficacy varies significantly across different tumor types. Histone deacetylase inhibitors (HDACi), such as SAHA, have demonstrated antitumor activity, but TNBC responds poorly to HDACi monotherapy, possibly due to feedback activation of the JAK-STAT pathway. Exploring the synergistic potential and underlying mechanisms of combining metformin with HDACi in TNBC treatment is crucial.

Methods: We predicted the synergistic effects of metformin and SAHA in TNBC using multiple computational methods (CMap, DTsyn, and DrugComb). We also developed a cancer-specific compound mimic library (CDTSL) and applied a three-step strategy to identify genes fitting the "metformin sensitization" model. Subsequently, we evaluated the synergistic effects of metformin and SAHA in TNBC cell lines through cell proliferation, colony formation, and apoptosis assays. Furthermore, we investigated the molecular mechanisms of the combined treatment using techniques such as transcriptome sequencing, chromatin immunoprecipitation (ChIP), Western blotting, and measurement of extracellular acidification rate (ECAR). Additionally, we assessed the in vivo antitumor effects of the combined therapy in a nude mouse subcutaneous xenograft model.

Results: CMap, DTsyn, and DrugComb all predicted the synergistic effects of SAHA and metformin in TNBC. The screening results revealed that HDAC10 played a key role in metformin sensitization. We found that the combination of metformin and SAHA exhibited synergistic antitumor effects (combination index CI < 0.9) in TNBC cell lines. Mechanistically, metformin inhibited histone acetylation on FGFR4, thereby blocking the feedback activation of FGFR4 downstream pathways induced by SAHA. Furthermore, metformin interfered with the glycolysis process induced by SAHA, altering the metabolic reprogramming of tumor cells. In in vivo experiments, the combined treatment of metformin and SAHA significantly inhibited the growth of subcutaneous tumors in nude mice.

Conclusions: Metformin enhances the sensitivity of TNBC to HDAC inhibitors by blocking the FGFR4 pathway and interfering with metabolic reprogramming. When used in combination with SAHA, metformin exhibits synergistic antitumor effects. Our study provides a theoretical basis for the combined application of HDAC inhibitors and metformin, potentially offering a new strategy for the treatment of TNBC.

Background: Social interaction is crucial for mental health across animal species. Social experiences, especially in early-life stages, strongly influence brain function and social behavior later in life. Acute social isolation (SI) increases motivation to seek social interaction, but little is known about its underlying neuronal and circuitry mechanisms. Here, we focus on oxytocin signaling in the ventral tegmental area (VTA), a vital node of the brain's reward network, as a potential mechanism for SI-induced craving for social interaction.

Methods: Adolescent (4-week-old) or adult (14-week-old) male C57BL/6J mice underwent a 1-week SI. Free interaction, object exploration, three-chamber social approach, and habituation tests were used to assess social and non-social behavior changes. Viral vectors were used to decipher the underlying neural circuitry, and chemogenetic techniques were applied to modify neuronal activity.

Results: We found that in male C57BL/6J mice, SI during adolescence, but not adulthood, leads to increased craving for social interaction and object exploration, accompanied by impaired social habituation, social novelty preference, and social recognition memory (SRM). SI-induced craving for social interaction and SRM deficit is still observed upon regrouping. Through cell-type-specific manipulations with designer receptors exclusively activated by designer drugs (DREADD), we show that oxytocin neurons in the paraventricular nucleus of the hypothalamus (PVN) are crucial for SI-induced social behavior changes. Chemogenetic activation of PVN oxytocin neurons recapitulates social behavior changes observed in SI mice, whereas chemogenetic inhibition of oxytocin neurons prevents social behavior changes caused by SI. Moreover, we found that dopaminergic neurons in the VTA mediate SI-induced craving for social interaction through their projections to the medial prefrontal cortex (mPFC), but not to the nucleus accumbens. Injection of a specific oxytocin receptor antagonist L368,899 into the VTA or chemical lesions of dopaminergic axon terminals in the mPFC with local application of 6-hydroxydopamine ameliorates SI-induced social behavior changes.

Conclusions: These findings suggest that adolescent SI has enduring effects on social behaviors in male mice through an oxytocinergic modulation of the VTA-to-mPFC dopaminergic circuit activity.

Background: Animal studies established that the locus coeruleus (LC) plays important roles in sleep and wakefulness regulation. Whether it contributes to sleep variability in humans is not yet established. Here, we investigated if the in vivo activity of the LC is related to the variability in the quality of Rapid Eye Movement (REM) sleep.

Methods: We assessed the LC activity of 34 healthy younger (~ 22y) and 18 older (~ 61y) individuals engaged in bottom-up and top-down cognitive tasks using 7-Tesla functional Magnetic Resonance Imaging (fMRI). We further recorded their sleep electroencephalogram (EEG) to evaluate associations between LC fMRI measures and REM sleep EEG metrics.

Results: Theta oscillation energy during REM sleep was positively associated with LC response in the top-down task. In contrast, REM sleep theta energy was negatively associated with LC activity in older individuals during the bottom-up task. Importantly, sigma oscillations power immediately preceding a REM sleep episode was positively associated with LC activity in the top-down task.

Conclusions: LC activity during wakefulness was related to REM sleep intensity and to a transient EEG change preceding REM sleep, a feature causally related to LC activity in animal studies. The associations depend on the cognitive task, suggesting that a balanced level of LC tonic activity during wakefulness is required for optimal expression of REM sleep. The findings may have implications for the high prevalence of sleep complaints reported in aging and for disorders such as insomnia, Alzheimer's, and Parkinson's disease, for which the LC may play pivotal roles through sleep.