Genomics, Proteomics & Bioinformatics•2024•DOI: 10.1093/gpbjnl/qzad003
The monkeypox virus (mpox virus, MPXV) epidemic in 2022 has posed a significant public health risk. Yet, the evolutionary principles of MPXV remain largely unknown. Here, we examined the evolutionary patterns of protein sequences and codon usage in MPXV. We first demonstrated the signal of positive selection in OPG027, specifically in the Clade I lineage of MPXV. Subsequently, we discovered accelerated protein sequence evolution over time in the variants responsible for the 2022 outbreak. Furthermore, we showed strong epistasis between amino acid substitutions located in different genes. The codon adaptation index (CAI) analysis revealed that MPXV genes tended to use more non-preferred codons compared to human genes, and the CAI decreased over time and diverged between clades, with Clade I > IIa and IIb-A > IIb-B. While the decrease in fatality rate among the three groups aligned with the CAI pattern, it remains unclear whether this correlation was coincidental or if the deoptimization of codon usage in MPXV led to a reduction in fatality rates. This study sheds new light on the mechanisms that govern the evolution of MPXV in human populations.
Stem Cell Research & Therapy•2025•DOI: 10.1186/s13287-025-04525-w
Background Mesenchymal stem cells (MSCs) are widely used in treating autoimmune diseases. However, replicative senescence limits the quantity and quality of MSCs during population doublings in vitro. Transcription factor SOX4 is a crucial regulator of cell fate and stemness. This study aims to explore the role of SOX4 in senescence of MSCs and enhance their therapeutic efficacy in systemic lupus erythematosus (SLE). Methods In early-passage MSCs (P3), late-passage MSCs (P8), SOX4 downregulated P3-MSCs or SOX4 overexpressed P8-MSCs, cell morphology, mitochondrial reactive oxygen species (mtROS), senescence-associated β-galactosidase (SA-β-Gal) activity, cell proliferation rate, senescence-associated secretory phenotype (SASP) factors, cell cycle suppressors, the immunosuppressive effects on T cell activation and proliferation and the expression levels of SOX4 were determined. Imiquimod induced SLE mice were transplanted with P3-MSCs and P8-MSCs or control and SOX4 overexpressed P8-MSCs, and clinical symptoms were assessed. Results Compared to P3-MSCs, P8-MSCs display a senescent phenotype, increased mtROS, SA-β-Gal activity, SASP factors, and cell cycle suppressors p53, p21, and p16. Additionally, P8-MSCs have a reduced immunosuppressive function on T cell activation and proliferation, and express lower levels of SOX4. Downregulation of SOX4 in P3-MSCs promotes cellular senescence and impairs their immunosuppressive function. Conversely, overexpression of SOX4 in P8-MSCs ameliorates cellular senescence and enhances their immunosuppressive function. Furthermore, transplantation of P3-MSCs or SOX4-overexpressing P8-MSCs demonstrates greater therapeutic significantly efficacy in SLE mice compared to P8-MSCs.
Acta Biochimica et Biophysica Sinica•2025•DOI: 10.3724/abbs.2025016
CRISPR-Cas nucleases have been extensively used in molecular detections, especially highly sensitive nucleic acid detections. In these detections, Cas nucleases are programmed by the guide RNA to respond to the detection targets and cleave the chemically labelled molecular beacons by the trans-cleavage activity to produce the detection signal. To improve sensitivity, nucleic acid amplification technologies are usually introduced to give a pre-amplification of the nucleic acid targets, increasing the detection sensitivity extraordinarily. Polymerase chain reaction (PCR) technology has been used for pre-amplification in laboratories, and isothermal amplification technologies are applied to meet point-of-care testing (POCT) needs because they avoid the use of sophisticated thermal cycling devices. The recombinase polymerase amplification (RPA) technology that amplifies nucleic acid targets isothermally at 37–42°C has been combined with CRISPR-Cas nucleases to establish advantageous nucleic acid detection assays, e.g., the SHERLOCK, which combines with Cas13a, and the DETECTR, which combines with Cas12a. It has been challenging to integrate Cas nucleases and RPA in a one-pot reaction system because the cleavage activity of Cas stimulated by even small amounts of the targets can interfere with amplification by digesting the primers or the newly amplified fragments. Thus, many assays based on Cas nucleases and RPA are in a two-step setting, with pre-amplification and Cas cleavage being isolated as two independent procedures. The two-step setting ensures that amplification and cleavage occur under favorable conditions but sacrifices operational convenience and introduces the risk of cross-contamination. In efforts to establish one-pot RPA-Cas assays, many strategies have been applied, including the use of photocontrolled guide RNA to activate the Cas nuclease at a preferred timepoint, the use of a suboptimal protospacer adjacent motif (PAM) to suppress Cas activity, the generation of dynamic aqueous multiphase with sucrose or glycerol to partially separate the two reactions, and extensive optimization of the RPA-CRISPR reaction system to achieve a subtle balance between the two reactions. In this study, a one-pot CRISPR-Cas12b and RPA combined assay with a temperature switch (CRATS) was established utilizing the reaction temperature difference between RPA and CRISPR-Cas12b cleavage. The Cas12b used in this study, AaCas12b, is a type V-B CRISPR‒Cas nuclease from Alicyclobacillus acidiphilus. It has a bi-lobed architecture consisting of an α-helical recognition lobe containing the REC domains and a nuclease lobe containing the WED, RuvC and Nuc domains. As a dual-RNA-guided DNA endonuclease, Cas12b can be guided by a chimeric single-guide (sg) RNA, and its trans-cleavage activity is specifically activated by the DNA target and results in nonspecific cleavage of single-stranded (ss) DNA molecules, which can be used to produce detection signals if the ssDNA is appropriately labelled as the molecular beacon. As the temperature for RPA is 37°C and the trans-cleavage of Cas12b is active at 60°C, CRATS uses temperature switching to adjust the major on-going reaction in the one-pot system and realizes sequential amplification of the target and cleavage reactions for signal detection. The detection target of this study, monkeypox virus, is an infectious pathogen that has caused the announcement of the Public Health Emergency of International Concern (PHEIC) by the World Health Organization (WHO) twice in recent years. In this one-pot CRATS assay, the reaction reagents of CRISPR-Cas12b and RPA are mixed in a single tube. After the addition of the sample containing the detection target, the reaction was carried out at 37°C for 20 min for amplification, followed by 60°C for 20 min for Cas12b cleavage. The fluorescently labelled molecular beacon is cleaved by Cas12b to release the FAM fluorophore from quenching, producing a fluorescence signal that is visualized under blue light. CRATS shows a high sensitivity of 100 copies of the target DNA per reaction and good specificity, providing a novel strategy of temperature switching to integrate CRISPR-Cas and RPA in a one-pot reaction system. Moreover, it provides a POCT-friendly tool for the detection of the important infectious pathogen monkeypox virus.
Acta Biochimica et Biophysica Sinica•2025•DOI: 10.3724/abbs.2025019
Cancer cells evade immune detection through checkpoint molecules like PD-L1 and PD-L2 which suppress T-cell activation. While PD-L1 is well-studied, the role of PD-L2 remains unclear. Pyruvate kinase M2 (PKM2), a metabolic enzyme, influences immune checkpoint regulation, but its role in PD-L1 and PD-L2 modulation is not well defined. Here, we investigate the role of pyruvate kinase M2 (PKM2) in modulating the immune checkpoint molecules PD-L1 and PD-L2 via GATA3 in cancer cells, with insights from both human and mouse models. We find that PKM2 enhances PD-L1 expression while inhibiting PD-L2, a dual regulatory mechanism that facilitates immune evasion. Knockdown and overexpression experiments revealed GATA3 as a key mediator. PKM2 knockout reduced GATA3 level, leading to decreased PD-L1 and increased PD-L2 expression. Chromatin immunoprecipitation (ChIP)-qPCR demonstrates that GATA3 functions as a direct transcription factor capable of binding to the promoters of PD-L1 and PD-L2. In silico analyses of 81 esophageal squamous cell carcinoma (ESCC) cases from the TCGA database demonstrate that PKM2 mRNA is unrelated to PD-L1 and PD-L2 expression but is negatively correlated with CD8+ T-cell infiltration in ESCC. To further validate these findings, we establish a xenograft model using immune-competent C57/BL6N mice, where knockdown of PKM2 results in significant downregulation of both PD-L1 and PD-L2 expression. Collectively, these findings underscore the divergent roles of PKM2 in regulating immune checkpoint expression in human and mouse cancer models and suggest that targeting the PKM2-GATA3 axis could enhance cancer immunotherapy by fine-tuning PD-L1 and PD-L2 levels.
Acta Biochimica et Biophysica Sinica•2025•DOI: 10.3724/abbs.2025124
Preeclampsia (PE) involves complex metabolic-inflammatory interactions, yet the mechanistic links among glycolysis, protein lactylation, and pyroptosis in placental pathogenesis remain undefined. In this study, we explore their tripartite relationship with PE development by combining bioinformatics analysis of PE-associated transcriptomes with experimental validation using placental tissues from PE patients and healthy controls. To elucidate the underlying mechanism, we utilize in vitro models involving hypoxic endothelial cell cultures, pharmacological glycolysis inhibition via 2-deoxyglucose, and genetic modulation of hexokinase 2 (HK2) expressions through siRNA silencing and plasmid-based overexpression. Molecular profiling is used to assess the expressions of key glycolytic enzymes, lactylation markers, and pyroptosis-related factors. Compared with control placental tissues, PE placental tissues present significantly higher expressions of glycolytic enzymes, elevated protein lactylation levels, and increased pyroptosis markers. Similarly, hypoxic endothelial cells exhibit coordinated upregulation of these three pathways. Notably, pharmacological glycolysis inhibition significantly reduces both lactylation and pyroptosis levels. Genetic experiments further demonstrate that HK2 silencing decreases glycolytic activity, subsequently attenuating lactylation and pyroptosis, whereas HK2 overexpression has opposite effects, underscoring its central regulatory role in this metabolic-inflammatory axis. Collectively, these findings indicate that HK2-mediated glycolysis drives placental vascular endothelial lactylation and pyroptosis, revealing a novel mechanistic pathway in PE pathophysiology.
Acta Biochimica et Biophysica Sinica•2024•DOI: 10.3724/abbs.2024122
The main protease (Mpro) of coronaviruses plays a key role in viral replication, thus serving as a hot target for drug design. PF-00835231 is a promising inhibitor of SARS-CoV-2 Mpro. Here, we report the inhibitory potency of PF-00835231 against SARS-CoV-2 Mpro and seven Mpro mutants (G15S, M49I, Y54C, K90R, P132H, S46F, and V186F) from SARS-CoV-2 variants. The results confirm that PF-00835231 has broad-spectrum inhibition against various coronaviral Mpros. In addition, the crystal structures of SARS-CoV-2 Mpro, SARS-CoV Mpro, MERS-CoV Mpro, and seven SARS-CoV-2 Mpro mutants (G15S, M49I, Y54C, K90R, P132H, S46F, and V186F) in complex with PF-00835231 are solved. A detailed analysis of these structures reveals key determinants essential for inhibition and elucidates the binding modes of different coronaviral Mpros. Given the importance of the main protease for the treatment of coronaviral infection, structural insights into Mpro inhibition by PF-00835231 can accelerate the design of novel antivirals with broad-spectrum efficacy against different human coronaviruses.
Acta Biochimica et Biophysica Sinica•2024•DOI: 10.3724/abbs.2024062
Ubiquitin-conjugation enzyme E2C (UBE2C) is a crucial component of the ubiquitin-proteasome system that is involved in numerous cancers. In this study, we find that UBE2C expression is significantly increased in mouse embryos, a critical stage during skeletal muscle development. We further investigate the function of UBE2C in myogenesis. Knockdown of UBE2C inhibits C2C12 cell differentiation and decreases the expressions of MyoG and MyHC, while overexpression of UBE2C promotes C2C12 cell differentiation. Additionally, knockdown of UBE2C, specifically in the tibialis anterior muscle (TA), severely impedes muscle regeneration in vivo. Mechanistically, we show that UBE2C knockdown reduces the level of phosphorylated protein kinase B (p-Akt) and promotes the degradation of Akt. These findings suggest that UBE2C plays a critical role in myoblast differentiation and muscle regeneration and that UBE2C regulates myogenesis through the Akt signaling pathway.
Chinese Journal of Tissue Engineering Research•2026•DOI: 10.12307/2026.21447
BACKGROUND: Neobavaisoflavone could promote bone formation and may be a potential small molecule drug for bone regeneration. The use of 3D printed bone tissue engineering scaffolds as drug delivery carriers for neobavaisoflavone is expected to enhance the potential application of bone regeneration. OBJECTIVE: To explore the effects of polylactic acid/polydopamine/neobavaisoflavone bone scaffold on osteoclast and osteoblast activity. METHODS: (1) Fused deposition modeling technology was used to manufacture a 3D printed polylactic acid scaffold. These polylactic acid scaffolds were immersed in a dopamine solution containing or without neobavaisoflavone to produce polylactic acid/polydopamine/neobavaisoflavone scaffolds and polylactic acid/polydopamine scaffolds, respectively. The surface morphology, surface hardness, and compressive strength of the three groups of scaffolds were characterized, and the drug release properties of the polylactic acid/polydopamine/neobavaisoflavone scaffolds were investigated. (2) Mouse embryonic osteoblast MC3T3-E1 cells were co-cultured with the three groups of scaffolds. CCK-8 assay and live/dead staining were used to evaluate the cytocompatibility of the scaffolds. Transwell assay was used to evaluate the effect of scaffolds on osteoblast migration. Alkaline phosphatase quantitative assay and alizarin red staining were used to evaluate the effect of scaffolds on osteoblast differentiation. RAW264.7 cells were co-cultured with the three groups of scaffolds. After osteoclast induction, tartrate-resistant acid phosphatase staining was used to evaluate the effect of scaffolds on osteoclast differentiation. RESULTS AND CONCLUSION: (1) Scanning electron microscopy showed that all three groups of scaffolds had three-dimensional structure and regular interconnected porous structure with an average pore size of 400 µm. The surface hardness and compressive strength of polylactic acid/polydopamine scaffolds and polylactic acid/polydopamine/neobavaisoflavone scaffolds were higher than those of polylactic acid scaffolds (P < 0.05). Polylactic acid/polydopamine/neobavaisoflavone scaffolds had good drug release behavior and could continuously release drugs for more than 14 days in vitro. (2) CCK-8 assay and live/dead staining showed that all three groups of scaffolds had good cytocompatibility, and polylactic acid/polydopamine scaffolds and polylactic acid/polydopamine/neobavaisoflavone scaffolds could promote MC3T3-E1 cell proliferation. Transwell assay showed that compared with polylactic acid scaffolds, polylactic acid/polydopamine scaffolds and polylactic acid/polydopamine/neobavaisoflavone scaffolds could promote MC3T3-E1 cell migration. Alkaline phosphatase quantitative assay and alizarin red staining showed that compared with the other two groups, polylactic acid/polydopamine scaffolds and polylactic acid/polydopamine/neobavaisoflavone scaffolds could promote osteogenic differentiation of MC3T3-E1 cells. Tartrate-resistant acid phosphatase staining showed that polylactic acid/polydopamine/neobavaisoflavone scaffolds could inhibit osteoclast differentiation of RAW264.7 cells. (3) These results indicate that polylactic acid/polydopamine/neobavaisoflavone scaffolds have good biosafety and can promote bone regeneration by regulating osteoblast and osteoclast activities.
Chinese Journal of Tissue Engineering Research•2026•DOI: 10.12307/2026.21517
BACKGROUND: The sacro-femoro-pubic angle, as a coronal plane alternative to the pelvic tilt angle, has an unclear association in different Lenke classifications of adolescent idiopathic scoliosis, limiting clinical application of the sacro-femoro-pubic angle in classification-specific assessment. OBJECTIVE: To explore the correlation between the sacro-femoro-pubic angle and spine-pelvic parameters (pelvic incidence, pelvic tilt, sacral slope, lumbar lordosis, thoracic kyphosis, and main Cobb angle) in patients with Lenke types 1, 5, and 6 adolescent idiopathic scoliosis, and to establish classification-specific regression models. METHODS: Clinical and imaging data of 191 adolescent idiopathic scoliosis patients were retrospectively analyzed, including 60 with Lenke type 1, 65 with Lenke type 5, and 66 with Lenke type 6. Pearson or Spearman correlation analysis was used to assess the correlation between left and right sacro-femoro-pubic angles and pelvic incidence, pelvic tilt, sacral slope, lumbar lordosis, thoracic kyphosis, Cobb angle, and sagittal vertical axis. Statistically significant parameters were subjected to univariate linear regression analysis to construct linear regression models. RESULTS AND CONCLUSION: In Lenke type 1 patients, the right sacro-femoro-pubic angle was significantly negatively correlated with pelvic tilt (r=-0.366, P=0.028), and both left and right sacro-femoro-pubic angles were significantly positively correlated with lumbar lordosis (r=0.429, P=0.007; r=0.460, P=0.007). In Lenke type 5 patients, both left and right sacro-femoro-pubic angles were significantly positively correlated with lumbar lordosis (r=0.415, P=0.007; r=0.400, P=0.007). In Lenke type 6 patients, both left and right sacro-femoro-pubic angles were significantly negatively correlated with pelvic tilt (r=-0.385, P=0.007; r=-0.376, P=0.014). Univariate regression models quantified these associations (R²=0.13-0.21), with the largest slope for the sacro-femoro-pubic angle-lumbar lordosis regression in Lenke type 5 (0.85-0.89), and slopes of -0.52 to -0.54 for the sacro-femoro-pubic angle-pelvic tilt regression in Lenke type 6. These findings indicate that the associations between sacro-femoro-pubic angle and spine-pelvic parameters are classification-specific: Lenke types 1 and 5 predominantly show positive correlations with lumbar lordosis, while Lenke type 6 is characterized by bilateral negative correlations with pelvic tilt.
Stem Cell Research & Therapy•2026•DOI: 10.1186/s13287-026-05100-7
Background: Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) hold therapeutic potential for spinal cord injury (SCI), yet their mechanisms remain unclear. We hypothesized that investigating in situ transcriptional reprogramming of transplanted hUC-MSCs within the spinal cord microenvironment (SCE) could identify crucial genes for SCI repair. Methods: DiD-labeled hUC-MSCs were intrathecally transplanted in rats with or without sub-acute spinal cord bilateral hemisection injury and retrieved for RNA-seq. Comparative transcriptomic analysis and functional screenings in vitro and in vivo, including heterologous synapse formation assay, transplantation of MSCs with gene overexpression or knockdown, AAV-mediated neuron-specific gene expression in SCI rats, behavioral tests, and motor evoked potentials (MEPs), identified Neuroligin 3 (Nlgn3) as a novel target. Immunoprecipitation-mass spectrometry (IP-Mass spec), cell aggregation assay, and immuno-electron microscopy revealed functional interacting partners. RT-qPCR, western blotting, immunofluorescence, and co-IP elucidated mechanisms. Results: NLGN3, a neuronal cell adhesion molecule activated by SCE in transplanted hUC-MSCs, promoted therapeutic efficacy. Neuron-specific restoration of Nlgn3 in injured spinal cord alone achieved comparable therapeutic effects. Mechanistically, Nlgn3 recruits synaptic vesicle proteins Sar1a and Hspa8 to modulate synaptic strength. Combinatorial restoration of Nlgn3 with Sar1a or Hspa8 synergistically enhanced SCI repair. Conclusions: This work unveils a novel therapeutic role for Nlgn3 in SCI, enhancing MSC transplantation efficacy and directly promoting neural circuit reconstruction.
Acta Biochimica et Biophysica Sinica•2026•DOI: 10.3724/abbs.2026080
DNAJC9, an HSP40 family member with histone chaperone function, exhibits unclear roles in cervical cancer. DNAJC9 is specifically overexpressed in malignant cervical cancer cells, and downregulation of DNAJC9 inhibits proliferation, induces G1/S arrest, and suppresses tumorigenicity. GLI1 has been identified as a key downstream effector of DNAJC9, and GLI1 rescue reverses proliferation defects. Mechanistically, DNAJC9 promotes the p300-H3 interaction to sustain H3K27ac at the GLI1 enhancer and facilitate GLI1 transcription, driving proliferation. Furthermore, DNAJC9 expression correlates positively with GLI1 in clinical specimens, suggesting that the DNAJC9-GLI1 axis is a potential prognostic marker and therapeutic target.
Acta Biochimica et Biophysica Sinica•2025•DOI: 10.3724/abbs.2025124
Preeclampsia (PE) is a multisystem syndrome affecting 2–8% of pregnancies, with placental dysfunction as a central driver. Metabolic dysregulation, particularly aberrant glycolysis, has been implicated in PE pathogenesis, but the mechanistic links among glycolysis, protein lactylation, and pyroptosis remain undefined. This study combined bioinformatics analysis of PE-associated transcriptomes with experimental validation using placental tissues from PE patients and healthy controls. In vitro models employed hypoxic endothelial cell cultures, pharmacological glycolysis inhibition via 2-deoxyglucose, and genetic modulation of hexokinase 2 (HK2) through siRNA silencing and plasmid-based overexpression. Molecular profiling assessed key glycolytic enzymes, lactylation markers, and pyroptosis-related factors. PE placental tissues exhibited significantly higher expressions of glycolytic enzymes, elevated protein lactylation, and increased pyroptosis markers compared to controls. Hypoxic endothelial cells showed coordinated upregulation of these pathways. Pharmacological glycolysis inhibition significantly reduced lactylation and pyroptosis. HK2 silencing decreased glycolytic activity, attenuating lactylation and pyroptosis, while HK2 overexpression had opposite effects, underscoring its central regulatory role. These findings indicate that HK2-mediated glycolysis drives placental vascular endothelial lactylation and pyroptosis, revealing a novel mechanistic pathway in PE pathophysiology and identifying HK2 as a potential therapeutic target.