Macrophages are innate immune cells with a dynamic range of reversible activation states including the classical pro-inflammatory (M1) and alternative anti-inflammatory (M2) states. Deciphering how macrophages regulate their transition from one state to the other is key for a deeper understanding of inflammatory diseases and relevant therapies. Common regulatory motifs reported for macrophage transitions, such as positive or double-negative feedback loops, exhibit a switchlike behavior, suggesting the bistability of the system. In this review, we explore the evidence for multistability (including bistability) in macrophage activation pathways at four molecular levels. First, a decision-making module in signal transduction includes mutual inhibitory interactions between M1 (STAT1, NF-KB/p50-p65) and M2 (STAT3, NF-KB/p50-p50) signaling pathways. Second, a switchlike behavior at the gene expression level includes complex network motifs of transcription factors and miRNAs. Third, these changes impact metabolic gene expression, leading to switches in energy production, NADPH and ROS production, TCA cycle functionality, biosynthesis, and nitrogen metabolism. Fourth, metabolic changes are monitored by metabolic sensors coupled to AMPK and mTOR activity to provide stability by maintaining signals promoting M1 or M2 activation. In conclusion, we identify bistability hubs as promising therapeutic targets for reverting or blocking macrophage transitions through modulation of the metabolic environment.

Osteosarcoma is the most frequent malignant disease of the bone. On the basis of early clinical experience in the 1960s with H-1 protoparvovirus (H-1PV) in osteosarcoma patients, this effective oncolytic virus was selected for systematic preclinical testing on various osteosarcoma cell cultures. A panel of five human osteosarcoma cell lines (CAL 72, H-OS, MG-63, SaOS-2, U-2OS) was tested. Virus oncoselectivity was confirmed by infecting non-malignant human neonatal fibroblasts and osteoblasts used as culture models of non-transformed mesenchymal cells. H-1PV was found to enter osteosarcoma cells and to induce viral DNA replication, transcription of viral genes, and translation to viral proteins. After H-1PV infection, release of infectious viral particles from osteosarcoma cells into the supernatant indicated successful viral assembly and egress. Crystal violet staining revealed progressive cytomorphological changes in all osteosarcoma cell lines. Infection of osteosarcoma cell lines with the standard H-1PV caused an arrest of the cell cycle in the G2 phase, and these lines had a limited capacity for standard H-1PV virus replication. The cytotoxicity of wild-type H-1PV virus towards osteosarcoma cells was compared in vitro with that of two variants, Del H-1PV and DM H-1PV, previously described as fitness variants displaying higher infectivity and spreading in human transformed cell lines of different origins. Surprisingly, wild-type H-1PV displayed the strongest cytostatic and cytotoxic effects in this analysis and thus seems the most promising for the next preclinical validation steps in vivo.

Single nucleotide changes were introduced into the non-structural (NS) coding sequence of the H-1 parvovirus (PV) infectious molecular clone and the corresponding virus stocks produced, thereby generating H1-PM-I, H1-PM-II, H1-PM-III, and H1-DM. The effects of the mutations on viral fitness were analyzed. Because of the overlapping sequences of NS1 and NS2, the mutations affected either NS2 (H1-PM-II, -III) or both NS1 and NS2 proteins (H1-PM-I, H1-DM). Our results show key benefits of PM-I, PM-II, and DM mutations with regard to the fitness of the virus stocks produced. Indeed, these mutants displayed a higher production of infectious virus in different cell cultures and better spreading capacity than the wild-type virus. This correlated with a decreased particle-to-infectivity (P/I) ratio and stimulation of an early step(s) of the viral cycle prior to viral DNA replication, namely, cell binding and internalization. These mutations also enhance the transduction efficiency of H-1PV-based vectors. In contrast, the PM-III mutation, which affects NS2 at a position downstream of the sequence deleted in Del H-1PV, impaired virus replication and spreading. We hypothesize that the NS2 protein—modified in H1-PM-I, H1-PM-II, and H1-DM—may result in the stimulation of some maturation step(s) of the capsid and facilitate virus entry into subsequently infected cells.

Graphical YloB in Solibacillus silvestris is not involved in calcium detoxification or biomineralization but is important for sporulation, likely transporting Ca²⁺ into the forespore for Ca-DPA accumulation and spore maturation.

Background: Genomic instability, a hallmark of cancer, leads to copy number variations disrupting gene dosage balance and contributing to tumor progression. One of the most affected oncogenes is MYC, whose overexpression is tightly regulated to avoid cytotoxicity. In aneuploid cancer cells, gene dosage compensation mechanisms involving microRNAs (miRNAs) from the miR-17/92 cluster contribute in regulating MYC expression. Targeting this miRNA-mediated compensation system represents a promising therapeutic strategy leading to an uncontrolled and lethal MYC overexpression. Results: Synthetic miRNA sponges targeting miR-17, miR-19a, and miR-20a, key regulators of MYC dosage compensation, were designed and validated. Breast cancer cells (MCF7) with stable exogenous MYC overexpression were used to assess the impact of sponge constructs on MYC regulation. Quantitative RT-PCR revealed a significant reduction in miRNA expression and a corresponding increase in endogenous MYC levels upon sponge treatment. Functional assays in multiple colorectal cancer cell lines with varying MYC copy numbers demonstrated a time-dependent increase in cell death following sponge transfection. Cytotoxic effects increased with MYC copy number, confirming a correlation between gene dosage sensitivity and therapeutic response. Conclusions: Our findings demonstrate that miRNA sponges targeting the miR-17/92 cluster can effectively disrupt MYC dosage compensation, leading to selective cytotoxicity in MYC-amplified cancer cells.

The rapid expansion of individualized treatment strategies necessitates advanced patient-specific screening platforms recapitulating tumor complexity. Here, we present such a platform preserving heterotypic cellular interactions and three-dimensional architecture which are both critical for predicting therapeutic responses. This cost-effective and versatile platform allows the rapid generation and functional testing of spheroids derived from primary human glioblastoma specimens. To maximize accessibility and ease of integration, we took advantage of commercially available kits to optimize the protocol of spheroid generation we had previously established. This enabled the robust, adaptable, and reproducible formation of homogeneous, multicellular spheroids within a few days. Spheroids generated from one patient specimen were structurally stable and showed a high degree of homogeneity over time. Immunohistochemical and flow cytometric analyses further revealed patient-specific cellular heterogeneity, underscoring the platform’s ability to preserve clinically relevant tumor features. Functionally, we demonstrate the applicability of this system for drug response profiling by assessing invasion dynamics following treatment with clinically relevant compounds. Collectively, our results establish a scalable and adaptable 3D screening platform that enables rapid, patient-specific phenotypic and functional analyses. This approach provides a powerful tool to complement existing clinical workflows and holds promise for improving the prediction of therapeutic responses in glioblastoma.