Monopolar spindle 1 kinase (Mps1, also known as TTK protein kinase) inhibitors exert marked anticancer effects against triple‑negative breast cancer (TNBC) by causing genomic instability and cell death. As aneuploid cells are vulnerable to compounds that induce energy stress through adenosine monophosphate‑activated protein kinase (AMPK) activation, the synergistic effect of Mps1/TTK inhibition and AMPK activation was investigated in the present study. The combined effects of CFI‑402257, an Mps1/TTK inhibitor, and AICAR, an AMPK agonist, were evaluated in terms of cytotoxicity, cell‑cycle distribution, and in vivo xenograft models. Additional molecular mechanistic studies were conducted to elucidate the mechanisms underlying apoptosis and autophagic cell death. The combination of CFI‑402257 and AICAR showed selective cytotoxicity in a TNBC cell line. The formation of polyploid cells was attenuated, and apoptosis was increased by the combination treatment, which also induced autophagy through dual inhibition of the PI3K/Akt/mTOR and mitogen‑activated protein kinase (MAPK) signaling pathways. Additionally, the combination therapy showed strongly improved efficacy in comparison with CFI‑402257 and AICAR monotherapy in the MDA‑MB‑231 xenograft model. The present study suggested that the combination of CFI‑402257 and AICAR is a promising therapeutic strategy for TNBC.

GDM, as a metabolic disease during pregnancy, regulates GLUT3 translocation by AMPK, thereby affecting glucose uptake in trophoblasts. It provides a new research idea and therapeutic target for alleviating intrauterine hyperglycemia in GDM. STZ was used to construct GDM mice, inject AICAR into pregnant mice, and observe fetal and placental weight; flow cytometry was employed for the detection of glucose uptake by primary trophoblast cells; immunofluorescence was applied to detect the localization of GLUT3 and AMPK in placental tissue; Cocofal microscope was used to detect the localization of GLUT3 in trophoblast cells;qRT-PCR and Western blot experiments were carried out to detect the expression levels of GLUT3 and AMPK in placental tissue; CO-IP was utilized to detect the interaction of GLUT3 and AMPK. Compared with the normal pregnancy group, the weight of the fetus and placenta of GDM mice increased (P < 0.001), and the ability of trophoblasts to take up glucose decreased (P < 0.001). In addition, AMPK activity in trophoblasts and membrane localization of GLUT3 in GDM mice were down-regulated compared with normal pregnant mice (P < 0.05). There is an interaction between GLUT3 and AMPK. Activating AMPK in trophoblasts can up-regulate the expression of GLUT3 membrane protein in trophoblasts of mice (P < 0.05) and increase the glucose uptake of trophoblasts (P < 0.05). We speculate that inhibition of AMPK activity in GDM mice results in aberrant localization of GLUT3, which in turn attenuates glucose uptake by placental trophoblast cells. AICAR activates AMPK to increase the membrane localization of GLUT3 and improve the glucose uptake capacity of trophoblasts.

The emergence of RNA on the early Earth is likely to have been influenced by chemical and physical processes that acted to filter out various alternative nucleic acids. For example, UV photostability is thought to have favored the survival of the canonical nucleotides. In a recent proposal for the prebiotic synthesis of the building blocks of RNA, ribonucleotides share a common pathway with arabino- and threo-nucleotides. We have therefore investigated non-templated primer extension with 2-aminoimidazole-activated forms of these alternative nucleotides to see if the synthesis of the first oligonucleotides might have been biased in favor of RNA. We show that non-templated primer extension occurs predominantly through 5\'-5\' imidazolium-bridged dinucleotides, echoing the mechanism of template-directed primer extension. Ribo- and arabino-nucleotides exhibited comparable rates and yields of non-templated primer extension, whereas threo-nucleotides showed lower reactivity. Competition experiments confirmed the bias against the incorporation of threo-nucleotides. The incorporation of an arabino-nucleotide at the end of the primer acts as a chain terminator and blocks subsequent extension. These biases, coupled with potentially selective prebiotic synthesis, and the templated copying that is known to favour the incorporation of ribonucleotides, provide a plausible model for the effective exclusion of arabino- and threo-nucleotides from primordial oligonucleotides.

BACKGROUND: Simvastatin (Sim), a hydroxy-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, has been widely used in prevention and treatment of cardiovascular diseases. Studies have suggested that Sim exerts anti-fibrotic effects by interfering fibroblast proliferation and collagen synthesis. This study was to determine whether Sim could alleviate silica-induced pulmonary fibrosis and explore the underlying mechanisms.
METHODS: The rat model of silicosis was established by the tracheal perfusion method and treated with Sim (5 or 10 mg/kg), AICAR (an AMPK agonist), and apocynin (a NOX inhibitor) for 28 days. Lung tissues were collected for further analyses including pathological histology, inflammatory response, oxidative stress, epithelial mesenchymal transformation (EMT), and the AMPK-NOX pathway.
RESULTS: Sim significantly reduced silica-induced pulmonary inflammation and fibrosis at 28 days after administration. Sim could reduce the levels of interleukin (IL)-1β, IL-6, tumor necrosis factor-α and transforming growth factor-β1 in lung tissues. The expressions of hydroxyproline, α-SMA and vimentin were down-regulated, while E-cad was increased in Sim-treated rats. In addition, NOX4, p22pox, p40phox, p-p47phox/p47phox expressions and ROS levels were all increased, whereas p-AMPK/AMPK was decreased in silica-induced rats. Sim or AICAR treatment could notably reverse the decrease of AMPK activity and increase of NOX activity induced by silica. Apocynin treatment exhibited similar protective effects to Sim, including down-regulating of oxidative stress and inhibition of the EMT process and inflammatory reactions.
CONCLUSIONS: Sim attenuates silica-induced pulmonary inflammation and fibrosis by downregulating EMT and oxidative stress through the AMPK-NOX pathway.

We report the generation of ∼8 nm dual in-plane pores fabricated in a thermoplastic via nanoimprint lithography (NIL). These pores were connected in series with nanochannels, one of which served as a flight tube to allow the identification of single molecules based on their molecular-dependent apparent mobilities (i.e., dual in-plane nanopore sensor). Two different thermoplastics were investigated including poly(methyl methacrylate), PMMA, and cyclic olefin polymer, COP, as the substrate for the sensor both of which were sealed using a low glass transition cover plate (cyclic olefin co-polymer, COC) that could be thermally fusion bonded to the PMMA or COP substrate at a temperature minimizing nanostructure deformation. Unique to these dual in-plane nanopore sensors was two pores flanking each side of the nanometer flight tube (50 × 50 nm, width × depth) that was 10 μm in length. The utility of this dual in-plane nanopore sensor was evaluated to not only detect, but also identify single ribonucleotide monophosphates (rNMPs) by using the travel time (time-of-flight, ToF), the resistive pulse event amplitude, and the dwell time. In spite of the relatively large size of these in-plane pores (∼8 nm effective diameter), we could detect via resistive pulse sensing (RPS) single rNMP molecules at a mass load of 3.9 fg, which was ascribed to the unique structural features of the nanofluidic network and the use of a thermoplastic with low relative dielectric constants, which resulted in a low RMS noise level in the open pore current. Our data indicated that the identification accuracy of individual rNMPs was high, which was ascribed to an improved chromatographic contribution to the nano-electrophoresis apparent mobility. With the ToF data only, the identification accuracy was 98.3%. However, when incorporating the resistive pulse sensing event amplitude and dwell time in conjunction with the ToF and analyzed via principal component analysis (PCA), the identification accuracy reached 100%. These findings pave the way for the realization of a novel chip-based single-molecule RNA sequencing technology.

Bronchial thermoplasty (BT), an effective treatment for severe asthma, requires heat to reach the airway to reduce the mass of airway smooth muscle cells (ASMCs). Autophagy is involved in the pathological process of airway remodeling in patients with asthma. However, it remains unclear whether autophagy participates in controlling airway remodeling induced by BT. In this study, we aim to elucidate the autophagy-mediated molecular mechanisms in BT. Our study reveal that the number of autophagosomes and the level of alpha-smooth muscle actin (α-SMA) fluorescence are significantly decreased in airway biopsy tissues after BT. As the temperature increased, BT causes a decrease in cell proliferation and a concomitant increase in the apoptosis of human airway smooth muscle cells (HASMCs). Furthermore, increase in temperature significantly downregulates cellular autophagy, autophagosome accumulation, the LC3II/LC3I ratio, and Beclin-1 expression, upregulates p62 expression, and inhibits the AMPK/mTOR pathway. Furthermore, cotreatment with AICAR (an AMPK agonist) or RAPA (an mTOR antagonist) abolishes the inhibition of autophagy and attenuates the increase in the apoptosis rate of HASMCs induced by the thermal effect. Therefore, we conclude that BT decreases airway remodeling by blocking autophagy induced by the AMPK/mTOR signaling pathway in HASMCs.

Ribonucleotides represent the most common non-canonical nucleotides found in eukaryotic genomes. The sources of chromosome-embedded ribonucleotides and the mechanisms by which unrepaired rNMPs trigger genome instability and human pathologies are not fully understood. The available sequencing technologies only allow to indirectly deduce the genomic location of rNMPs. Oxford Nanopore Technologies (ONT) may overcome such limitation, revealing the sites of rNMPs incorporation in genomic DNA directly from raw sequencing signals. We synthesized two types of DNA molecules containing rNMPs at known or random positions and we developed data analysis pipelines for DNA-embedded ribonucleotides detection by ONT. We report that ONT can identify all four ribonucleotides incorporated in DNA by capturing rNMPs-specific alterations in nucleotide alignment features, current intensity, and dwell time. We propose that ONT may be successfully employed to directly map rNMPs in genomic DNA and we suggest a strategy to build an ad hoc basecaller to analyse native genomes.

Human DNA ligase 1 (LIG1) is the main replicative ligase that seals Okazaki fragments during nuclear replication and finalizes DNA repair pathways by joining DNA ends of the broken strand breaks in the three steps of the ligation reaction. LIG1 can tolerate the RNA strand upstream of the nick, yet an atomic insight into the sugar discrimination mechanism by LIG1 against a ribonucleotide at the 3\'-terminus of nick DNA is unknown. Here, we determined X-ray structures of LIG1/3\'-RNA-DNA hybrids and captured the ligase during pre- and post-step 3 the ligation reaction. Furthermore, the overlays of 3\'-rA:T and 3\'-rG:C step 3 structures with step 2 structures of canonical 3\'-dA:T and 3\'-dG:C uncover a network of LIG1/DNA interactions through Asp570 and Arg871 side chains with 2\'-OH of the ribose at nick showing a final phosphodiester bond formation and the other ligase active site residues surrounding the AMP site. Finally, we demonstrated that LIG1 can ligate the nick DNA substrates with pre-inserted 3\'-ribonucleotides as efficiently as Watson-Crick base-paired ends in vitro. Together, our findings uncover a novel atomic insight into a lack of sugar discrimination by LIG1 and the impact of improper sugar on the nick sealing of ribonucleotides at the last step of DNA replication and repair.

Toxin-antitoxin systems are preserved by nearly every prokaryote. The type II toxin MazF acts as a sequence-specific endoribonuclease, cleaving ribonucleotides at specific sequences that vary from three to seven bases, as has been reported in different host organisms to date. The present study characterized the MazEF module (MazEF-sth) conserved in the Symbiobacterium thermophilum IAM14863 strain, a Gram-negative syntrophic bacterium that can be supported by co-culture with multiple bacteria, including Bacillus subtilis. Based on a method combining massive parallel sequencing and the fluorometric assay, MazF-sth was determined to cleave ribonucleotides at the UACAUA motif, which is markedly similar to the motifs recognized by MazF from B. subtilis (MazF-bs), and by several MazFs from Gram-positive bacteria. MazF-sth, with mutations at conserved amino acid residues Arg29 and Thr52, lost most ribonuclease activity, indicating that these residues that are crucial for MazF-bs also play significant roles in MazF-sth catalysis. Further, cross-neutralization between MazF-sth and the non-cognate MazE-bs was discovered, and herein, the neutralization mechanism is discussed based on a protein-structure simulation via AlphaFold2 and multiple sequence alignment. The conflict between the high homology shared by these MazF amino acid sequences and the few genetic correlations among their host organisms may provide evidence of horizontal gene transfer.

The savory or umami taste of the amino acid glutamate is synergistically enhanced by the addition of the purines inosine 5\'-monophosphate (IMP) and guanosine 5\'-monophosphate (GMP) disodium salt. We hypothesized that the addition of purinergic ribonucleotides, along with the pyrimidine ribonucleotides, would decrease the absolute detection threshold of (increase sensitivity to) l-glutamic acid potassium salt (MPG). To test this, we measured both the absolute detection threshold of MPG alone and with a background level (3 mM) of 5 different 5\'-ribonucleotides. The addition of the 3 purines IMP, GMP, and adenosine 5\'-monophosphate (AMP) lowered the MPG threshold in all participants (P < 0.001), indicating they are positive modulators or enhancers of glutamate taste. The average detection threshold of MPG was 2.08 mM, and with the addition of IMP, the threshold was decreased by approximately 1.5 orders of magnitude to 0.046 mM. In contrast to the purines, the pyrimidines uridine 5\'-monophosphate (UMP) and cytidine 5\'-monophosphate (CMP) yielded different results. CMP reliably raised glutamate thresholds in 10 of 17 subjects, suggesting it is a negative modulator or diminisher of glutamate taste for them. The rank order of effects on increasing sensitivity to glutamate was IMP > GMP> AMP >> UMP// CMP. These data confirm that ribonucleotides are modulators of glutamate taste, with purines enhancing sensitivity and pyrimidines displaying variable and even negative modulatory effects. Our ability to detect the co-occurrence of glutamate and purines is meaningful as both are relatively high in evolutionarily important sources of nutrition, such as insects and fermented foods.