BMC Molecular and Cell Biology最新文献

The Cell Biology of Dictyostelium article collection highlights the benefits of using Dictyostelium as a model system for studying fundamental cellular processes. The studies compiled in this collection showcase the many ways Dictyostelium is used to enhance our understanding of the eukaryotic cell.

Triple-negative breast cancer (TNBC) presents a significant therapeutic challenge due to its aggressive nature, lack of hormone receptors, and limited targeted treatment options. The complexity of the disease is further compounded by mutations in high-penetrance genes such as BRCA1, BRCA2, and BAX, along with other apoptotic genes involved in tumorigenesis, apoptosis, and drug resistance. Targeting these genes through innovative therapeutic approaches is crucial for improving treatment outcomes. This in-silico study explores the potential of phytochemicals as natural, multi-targeted therapeutic agents against high-penetrance and apoptotic genes implicated in TNBC. Using the IMPPAT 2.0 database, 300 phytochemicals were systematically screened based on their pharmacokinetic properties and toxicity profiles to identify promising candidates. Among them, Bayogenin exhibited strong binding to BRCA2 (-9.3 kcal/mol) and PALB2 (-8.7 kcal/mol), surpassing the FDA-approved drug Olaparib in molecular docking studies. Molecular dynamics simulations over 200 ns further confirmed the stability of these phytochemical-protein complexes, showing consistent root mean square deviation, hydrogen bonding, and free energy profiles. These findings highlight the therapeutic potential of phytochemicals and their possible advantages over existing TNBC treatments. By targeting key molecular pathways, this study provides insights into the development of natural, multi-targeted therapeutic strategies, emphasizing their translational relevance for TNBC therapy.

Background: Astrocyte cilia are largely understudied due to the lack of available tools. Astrocyte research advanced with the establishment of Aldh1l1-Cre^ERT2, an inducible Cre line that specifically targets the astrocyte lineage. Here, we develop and compare genetic models that label astrocyte cilia in the developing prefrontal cortex (PFC) using Aldh1l1-Cre^ERT2 and Cre-dependent cilia reporters. We evaluate these models by testing different tamoxifen-induction protocols and quantifying the percentage of astrocytes labeled with the cilia reporters.

Results: We show that tamoxifen dosage impacts the expression of cilia reporters in astrocytes. We uncover the maximum cilia-labeling efficiency using recombined, constitutively-expressed cilia reporters.

Conclusion: The data reveal that only a subset of SOX9- positive astrocytes in the PFC possess cilia throughout development. Our work highlights the utility of Cre-Lox systems to target specific cell types and the importance of carefully validating genetic models.

Background: Antibodies against non-HLA antigens, such as MICA and MICB, have emerged as potential contributors to antibody-mediated rejection and graft failure. While MICA antibodies are well characterized, MICB-specific antibodies remain poorly understood due to the lack of standardized detection tools. To address this gap, we aimed to develop a cell-based platform expressing individual MICB antigens to evaluate the feasibility of detecting allele-specific anti-MICB antibodies in pre-kidney transplant sera.

Methods: HLA class I, MICA, and MICB-null human embryonic kidney (HEK)-293T cells were previously generated by CRISPR/Cas9. We established five cell lines expressing single MICB antigens (each MICB*002, *003, *004, *005:02, and *008 allele). A total of 64 pre-kidney transplant sera were tested to assess anti-MICB antibody responses to the five cell lines using flow cytometry.

Results: We successfully established and validated five HEK-293T cell lines each expressing a single MICB antigen using anti-MICB monoclonal antibody staining. No anti-MICB antibodies were detected in any of the 64 pre-transplant sera tested. This finding may reflect a low incidence of sensitization to MICB in this patient population and suggests the need for larger, more diverse cohorts in future studies to fully assess the prevalence of anti-MICB responses. The established cell lines provide a promising tool for future investigation of allele-specific anti-MICB antibody responses.

Conclusions: While the present study did not detect allele-specific anti-MICB antibody responses, establishing HEK-293T cell lines expressing single MICB antigens represents a significant methodological advance. This platform enables the potential assessment of immune responses targeted to individual MICB allotypes, thus offering new avenues for the future study of MICB immunogenicity in transplantation settings.

Background: Spontaneous abortion (SA) is a complex reproductive disease that poses significant clinical challenge. Circular RNAs (circRNAs), a specific class of endogenous non-coding RNAs, hold significant potential for preclinical diagnosis and therapeutic interventions in various diseases. However, the precise roles of circRNAs in SA have yet to be fully elucidated.

Methods: In this study, we employed RNA sequencing and quantitative real-time polymerase chain reaction (qRT-PCR) to identify an upregulated circRNA, circFTO (hsa_circ_0005941), in the placental villi of SA patients. We conducted in vitro and in vivo experiments to ascertainthe functional significance of circFTO in trophoblast cell lines. The molecular mechanisms associated with circFTO were predicted using online databases and confirmed through RNA immunoprecipitation (RIP) assays, Western blotting, and rescue experiments.Additionally, Actinomycin D was employed to assess changes in the stability of target messenger RNA (mRNA) under different treatments. Furthermore, colorimetric examinations were used to evaluate the m6A methylation levels of trophoblast cells, and meRIP-qPCR assays confirmed the m6A modification of CCAR1 mRNA.

Results: Our findings revealed that circFTO was upregulated in the placenta of SA patients. Functionally, downregulating circFTO expression enhanced trophoblast cell proliferation, migration, and invasion. Conversely, overexpression of circFTO inhibited these functions in trophoblast cells. Trophoblast organoids derived from normal pregnancies exhibited reduced proliferation upon overexpression of circFTO. Bioinformatics prediction and subsequent experiments demonstrated that circFTO directly bound to and negatively regulated IGF2BP2. Reducing the level of IGF2BP2 partially restored the alterations in trophoblast function caused by circFTO knockdown. Colorimetric assay, RNA decay experiments, and meRIP-qRT-PCR analysis revealed that circFTO knockdown increased m6A methylation levels in CCAR1 mRNA, while circFTO overexpression decreased m6A methylation levels. This modification is known to play a crucial role in Zygotic gene activation.

Conclusions: Our study unveils the pivotal functions of circFTO within trophoblasts and elucidates a unique circFTO-IGF2BP2-CCAR1 axis, which may hold significant potential as diagnostic and therapeutic targets for the treatment of SA.

Clinical trial number: Not applicable.

Background: Adherent-invasive Escherichia coli (AIEC) is linked to intestinal inflammation in inflammatory bowel disease (IBD). Arthrospira platensis and Lactobacillus helveticus exhibit anti-inflammatory properties individually, yet their effects remain underexplored in IBD-associated inflammation. We aimed to investigate the anti-inflammatory potential of L. helveticus and the hydroalcoholic extract of A. platensis (HA-A. platensis) in Caco-2 cells inflamed by IBD-associated E. coli.

Methods: Caco-2 cells inflamed by a Crohn's disease (CD)-associated E. coli strain (MOI 10) were treated with HA-A. platensis (2 mg/mL) and/or L. helveticus (MOI 50) in live (LBC), heat-killed (HKC), or cell-free supernatant (CFS) forms. The anti-invasion/adhesion properties of L. helveticus and/or HA-A. platensis were investigated by assessing the CD-associated E. coli invasion/adhesion rate (%). Signaling molecules (NF-κB, STAT3, NOD2) were analyzed via qPCR to capture pathway activation dynamics, while cytokines (TNF-α, IL-1β, IL-8, IL-10) were quantified by ELISA to assess secreted functional proteins.

Results: HA-A. platensis reduced E. coli adhesion by 68% (P < 0.001) and completely inhibited invasion. L. helveticus (live form) decreased adhesion by 88% and invasion by 90%. Combined treatment showed synergistic effects, reducing adhesion by 89% and fully blocking invasion. HA-A. platensis downregulated STAT3 expression by 0.4-fold (P < 0.01), while L. helveticus (heat-killed form) reduced NF-κB by 0.51-fold (P < 0.05) and increased NOD2 by 1.8-fold (P < 0.01). Cytokine analysis revealed that HA-A. platensis decreased IL-1β by 0.61-fold (P < 0.001), and L. helveticus (heat-killed) reduced TNF-α (0.51-fold) and IL-8 (0.23-fold) while elevating anti-inflammatory IL-10 (4.39-fold; P < 0.001).

Conclusions: L. helveticus and HA-A. platensis synergistically inhibit CD-associated E. coli pathogenicity and modulate inflammatory responses in vitro. These findings highlight their potential as adjunctive therapies for CD, warranting further preclinical validation.

Background: The aging heart undergoes physiological changes, many of which are sex dependent and encompass differential responses to cardiac stress. However, much about the molecular changes that occur within the aging heart is still unknown. Thyroid hormone (TH) and the posttranslational modification O-GlcNAcylation (O-GlcNAc) are independently known to regulate cardiac function; therefore, we tested the hypothesis that TH disorders affect cardiac protein O-GlcNAcylation in aged hearts.

Results: We treated male and female 18-22 month-old aged C57BL/6 mice to create euthyroid, hypothyroid, or hyperthyroid states. Western blots and RT-qPCR from cardiac tissue were used to determine changes in global O-GlcNAc levels along with key regulatory proteins in the O-GlcNAcylation process. Immunoprecipitation and western blotting compared global O-GlcNAc changes to differences on an individual protein. We found increased total O-GlcNAc levels for female hypo- and hyperthyroid mice and male hyperthyroid mice compared to sex-matched euthyroid hearts, with no change for male hypothyroid mice. TH's O-GlcNAc effect on female mice appears heart specific as liver O-GlcNAc levels were unchanged. The proteins regulating O-GlcNAcylation also demonstrated sex differences. Female hyperthyroid mice had increased protein expression of the O-GlcNAc regulatory proteins GFAT 1, GFAT 2, and OGT, whereas the hyperthyroid male mice showed decreased expression for the regulatory protein OGA. The hypothyroid female mice had increased protein expression for OGT and NAGK, whereas the hypothyroid male mice showed increased protein expression for NAGK alone. Interestingly, the directional changes in these protein levels did not match RNA transcription. We further found O-GlcNAc levels of the mitochondrial thiolase protein ACAA2 diverged from global O-GlcNAc changes. ACAA2 was hyper O-GlcNAcylated in the female hypothyroid group and hypo O-GlcNAcylated in the male hyperthyroid group whereas there was no change in female hyperthyroid or male hypothyroid.

Conclusion: Protein O-GlcNAcylation is potentially an important mechanism whereby TH perturbations affect the aged heart. We found sex influences O-GlcNAc regulation, global O-GlcNAc levels, and O-GlcNAc protein specificity in response to thyroid hormone perturbations. Our results also suggest the changes in cardiac O-GlcNAc levels are not solely due to TH transcriptional regulation of key O-GlcNAc regulatory enzymes.

Background: Short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) is a ubiquitously expressed mitochondrial enzyme with a role in the degradation of fatty acids. Because the protein also is a negative regulator of insulin secretion in pancreatic β-cells, inactivating mutations in the SCHAD gene (HADH) cause congenital hyperinsulinism of infancy (CHI) and severe hypoglycemia. Here we sought to identify novel interaction partners of SCHAD that might be particularly relevant for the endocrine pancreas.

Results: Employing the SCHAD protein as bait, we performed yeast 2-hybrid screening of a cDNA library made from human islets of Langerhans. Surprisingly, the screening revealed the intermediate filament protein keratin 8 (K8) as a putative interaction partner of SCHAD with very high confidence. Previous reports have linked K8 to glucose homeostasis, and we confirmed the SCHAD interaction by co-immunoprecipitation in HEK293 cells. SCHAD and K8 expression were then characterized in the human β-cell model EndoC-βH1. By using proximity ligation assay, we demonstrated that stimulating the cells with a high level of glucose triggered a transient increase in the interaction. However, when studying knockout mice, we found that the loss of either K8 or SCHAD did not change the expression level of the other interaction partner. Still, when K8 knockout mice were challenged with a ketogenic diet, upregulation of SCHAD expression was blunted compared to the upregulation observed in wildtype littermates.

Conclusions: We propose that the SCHAD protein interacts with K8 in a way that might be relevant for proper functioning of the pancreatic β-cell. Whether the SCHAD-K8 interaction influences the phenotype of CHI remains to be demonstrated.

Background: HIV infection is mainly described by depletion of CD4⁺ T-cells; however, this not only occurs in infected cells, also arise in uninfected immunological cells through the bystander effect. Extrinsic cell death, in particular the Fas pathway has been studied in HIV extensively, and an expression increase in both its ligand and receptor has been reported, however the TRAIL pathway has been less explored in this context, and little has been relating to the immune activation characteristic of the disease. This study aims to examine the effect of HIV infection in the activation of TRAIL and Fas death pathways in CD3⁺ CD4⁺ T-cells and CD4⁺ CD14 + monocyte derived from people living with HIV (PLWHIV) and its correlation with immune activation biomarkers in cell surface and serum.

Results: Expression of TRAIL receptor DR5 in CD3⁺ CD4⁺ T-cells and CD14⁺ CD4⁺ monocytes from PLWHIV were significatively increased, almost two and five times more than CD3⁺ CD4⁺ T-cells and CD14⁺ CD4⁺ monocytes from HIV-negative controls; respectively. In PLWHIV, DR5 and CCR5 expression were positively and negatively associated with time of infection; respectively. Simultaneously, DR5 was associated positively with CXCR4 expression in CD3⁺ CD4⁺-T cells and CD4⁺ CD14⁺ monocytes as well as the significant increase of serum levels of IL-18 in PLWHIV. In CD3⁺ CD4⁺-T cells from HIV patients, the expression of CD38 was upregulated. Finally, in CD14⁺ CD4⁺ monocytes from PLWHIV, it was observed an increase in early apoptosis in response to recombinant TRAIL ligand, an effect that was not inhibited by caspase 8 blockade.

Conclusions: In PLWHIV before ART, the activation and regulation of TRAIL pathway shows to be an important regulator in cell depletion. The expression of TRAIL DR5 significantly increased in CD3⁺ CD4⁺-T cells and CD4⁺ CD14⁺ monocytes from PLWHIV; in the same way DR5 was positively correlated with time of infection, with CXCR4 expression and with the significant increase in serum levels of IL-18, making it an interesting target for future treatments and as a marker for HIV disease progression.

Background: Protein folding is a complex process in which amino acid sequences encode the information required for a polypeptide chain to fold into its functional three-dimensional (3D) structure. Many proteins share common substructures and recurring secondary structure elements that contribute to similar 3D folding patterns, even across different protein families. This study examines two distinct groups of proteins, the RNase A-like fold and the trypsin-like serine protease fold, classified by SCOPe. These proteins share only some substructures that contribute to their folding cores. Despite minimal sequence identity, they exhibit partial structural similarities in their 3D topologies. We used a sequence-based approach, including inter-residue average distance statistics and contact frequency prediction, to explore these folding characteristics. Structural observations guided further analyses of conserved hydrophobic residue packing, highlighting key folding units within each fold.

Results: Our analysis predicted two compact regions within each protein group. Interactions between these regions form a partially shared topology. We identified conserved hydrophobic residues critical to these interactions, suggesting a common mechanism for establishing these structural features. Despite overall structural differences between the RNase A-like and trypsin-like folds, our findings emphasize the presence of a shared partial folding core.

Conclusions: The partially shared structural features in the RNase A-like and trypsin-like serine protease folds reflect a convergent folding mechanism. This mechanism underscores the evolutionary adaptation of protein folding, where distinct folds can still retain critical, conserved structural motifs. These findings highlight how proteins with overall different topologies can evolve to share key folding features, demonstrating the elegance and efficiency of protein evolution.