Design, Synthesis and Anti-Lung Cancer Evaluation of 1, 2, 3-Triazole Tethered Dihydroartemisinin-Isatin Hybrids

A series of 1,2,3-triazole tethered dihydroartemisinin-isatin hybrids 8a-c and 9a-k were designed and synthesized. Their antiproliferative activity against A549, doxorubicin-resistant A549 (A549/DOX) as well as cisplatin-resistant A549 (A549/DDP) lung cancer cell lines was also investigated in this study. All hybrids (half maximal inhibitory concentration/IC50: 7.54–73.8 μM) were more potent than the parent drug dihydroartemisinin (IC50: 69.4–88.0 μM) and also non-cytotoxic towards mouse embryonic fibroblast cells NIH/3T3 (IC50: >100 μM). The structure-activity relationships illustrated that the substituents on C-3 and C-5 position of isatin moiety influenced the activity significantly. Imine at C-3 position decreased the activity, whereas fluoro at C-5 position enhanced the activity. In particular, hybrids 8a,c (IC50: 7.54–12.1 μM) and 9i (IC50: 9.10–15.9 μM) were comparable to cisplatin (IC50: 7.54–15.9 μM vs 9.38–19.7 μM) against A549 and A549/DOX, but 4.6–7.6 folds more potent than that of cisplatin (IC50: 8.77–14.3 μM vs 66.9 μM) against A549/DDP cells. Moreover, hybrids 8a,c exhibited excellent stability (liver microsomes: 68–83%) in mouse/human microsomes and good pharmacokinetic properties, demonstrating their potential as a novel anti-lung cancer chemotherapeutic candidates.

Subfamily-specific differential contribution of individual monomers and the tether sequence to mouse L1 promoter activity

BackgroundThe internal promoter in L1 5’UTR is critical for autonomous L1 transcription and initiating retrotransposition. Unlike the human genome, which features one contemporarily active subfamily, four subfamilies (A_I, Gf_I and Tf_I/II) have been amplifying in the mouse genome in the last one million years. Moreover, mouse L1 5’UTRs are organized into tandem repeats called monomers, which are separated from ORF1 by a tether domain. In this study, we aim to compare promoter activities across young mouse L1 subfamilies and investigate the contribution of individual monomers and the tether sequence.ResultsWe observed an inverse relationship between subfamily age and the average number of monomers among evolutionarily young mouse L1 subfamilies. The youngest subgroup (A_I and Tf_I/II) on average carry 3-4 monomers in the 5’UTR. Using a single-vector dual-luciferase reporter assay, we compared promoter activities across six L1 subfamilies (A_I/II, Gf_I and Tf_I/II/III) and established their antisense promoter activities in a mouse embryonic fibroblast cell line. Using consensus promoter sequences for three subfamilies (A_I, Gf_I and Tf_I), we dissected the differential roles of individual monomers and the tether domain in L1 promoter activity. We validated that, across multiple subfamilies, the second monomer consistently enhances the overall promoter activity. For individual promoter components, monomer 2 is consistently more active than the corresponding monomer 1 and/or the tether for each subfamily. Importantly, we revealed intricate interactions between monomer 2, monomer 1 and tether domains in a subfamily-specific manner. Furthermore, using three-monomer 5’UTRs, we established a complex nonlinear relationship between the length of the outmost monomer and the overall promoter activity.ConclusionsThe laboratory mouse is an important mammalian model system for human diseases as well as L1 biology. Our study extends previous findings and represents an important step toward a better understanding of the molecular mechanism controlling mouse L1 transcription as well as L1’s impact on development and disease.

Comparison of the somatic TADs and lampbrush chromomere-loop complexes in transcriptionally active prophase I oocytes

In diplotene oocyte nuclei of all vertebrate species, except mammals, chromosomes lack interchromosomal contacts and chromatin is linearly compartmentalized into distinct chromomere-loop complexes forming lampbrush chromosomes. However, the mechanisms underlying the formation of chromomere-loop complexes remain unexplored. Here we aimed to juxtapose somatic topologically associating domains (TADs), recently identified in chicken embryonic fibroblasts, with chromomere-loop complexes in lampbrush meiotic chromosomes. By measuring 3D-distances and colocalization between linear equidistantly located genomic loci, positioned within one TAD or separated by a TAD border, we confirmed the presence of predicted TADs in chicken embryonic fibroblast nuclei. Using three-colored FISH with BAC probes we mapped equidistant genomic regions included in several sequential somatic TADs on isolated chicken lampbrush chromosomes. Eight genomic regions, each comprising two or three somatic TADs, were mapped to non-overlapping neighboring lampbrush chromatin domains - lateral loops, chromomeres or chromomere-loop complexes. Genomic loci from the neighboring somatic TADs could localize in one lampbrush chromomere-loop complex, while genomic loci belonging to the same somatic TAD could be localized in neighboring lampbrush chromomere-loop domains. In addition, FISH-mapping of BAC probes to the nascent transcripts on the lateral loops indicates transcription of at least 17 protein-coding genes and 2 non-coding RNA genes during the lampbrush stage of chicken oogenesis, including genes involved in oocyte maturation and early embryo development.

Cell proliferative properties of Forcespinning® nopal composite nanofibers

In this study, Forcespinning® was used to produce nanofibers composed of Opuntia cochenillifera, “nopal,” mucilage (N) extract, chitosan (CH), and pullulan (PL) (N/CH/PL). These nopal-incorporating nanofibers were examined for their ability to sustain adhesion and proliferation of mouse embryonic fibroblast (NIH 3T3) cells. After a 6-day incubation period, N/CH/PL nanofibers displayed robust cell proliferation, with continued cell growth after an extended incubation period of 14 days. These results demonstrate that natural bioactive compounds can be combined with biodegradable polymers to provide an enhanced environment for cell growth, suggesting potential natural active ingredients as alternatives in wound dressings.

About 4-day Rhythm of Proliferative Activity of Fibroblast-like Cell Cultures Depends on the Environmental Factor

A study of the 4-day rhythm of the proliferative activity of the embryonic fibroblast-like cells in the logarithmic growth phase was carried out. It was shown that in cell cultures obtained on different days from embryos of different ages, the phase of the 4-day rhythm coincides. In vitro the maxima of the proliferative activity were consistent with the minima of the motor activity of mice. Freezing the culture for 2 or 6 days does not cause a shift in the phase of the 4-day rhythm of cell proliferative activity compare with the unfreezing culture. That indicates the existence of an external synchronizer, which determines the 4-day infradian rhythm of the proliferative activity of embryonic cells. Then we daily thawed samples of single L-929 culture of mice fibroblast-like cells for 22 and 17 days and researched the dynamics of its proliferative activity. We also showed 4-day rhythm of the simultaneous increase in the number of cells for all thawed samples. Taking into account that deep freezing of a culture leads to the cessation of all life processes, the fact we obtained indicates an exogenous mechanism of the formation of about a 4-day rhythm of the proliferative activity of cell culture.

CDK5 influences the organization of the Circadian Machinery in peripheral clocks

Circadian rhythms are self-sustained physiological changes that drive rhythmicity within the 24-hours cycles. Posttranslational modifications (PMTs), such as protein phosphorylation, acetylation, sumoylation, and ubiquitination, are biochemical processes that modify protein structure and functions, ensuring circadian rhythm precision. For example, phosphorylation is considered the most important hallmark of rhythmicity from cyanobacteria to mammals. Cyclin-dependent kinase 5 (CDK5) has been shown to regulate the mammalian SCN’s circadian clock via phosphorylation of PER2. Here, we show that CDK5 influences the clock machinery assembling, using immortalized mouse embryonic fibroblast as an in vitro model for studying the peripheral clock. In fact, the circadian period at the cellular level is lengthened. Furthermore, the clock-controlled gene’s expression amplitude is dampened in Cdk5 ko cell lines, while the phase is delayed about 4 hours.Taken together, we show in vitro that CDK5 is critically involved in regulating the peripheral clocks, influencing their temporal and spatial dynamics.

Acrylamide Induced Oxidative Cellular Senescence in Embryonic Fibroblast Cell Line (NIH 3T3): A Protection by Carvacrol

Background: Stress-induced cellular senescence is a perpetual state of cell cycle arrest occurring in proliferating cells in response to stressful conditions. It is believed that oxidative stress plays a unique role in this process. As a reactive chemical compound that can induce oxidative stress, acrylamide is widely applied in several fields. Carvacrol is a liquid phenolic monoterpenoid found in essential oils of some plants and is known for its antioxidant and anticarcinogenic properties. Objectives: The current study aimed to evaluate the effects of carvacrol on oxidative stress and cellular senescence induced by acrylamide in the NIH 3T3 cell line. Methods: NIH 3T3 embryonic fibroblast cells were exposed to different concentrations of acrylamide, carvacrol, and H2O2 in a cell culture medium. The level of β-galactosidase (SA-β-gal) activity, as a marker of cellular senescence, was measured using staining and quantitative assays. Furthermore, to measure oxidative stress parameters, the content of glutathione and lipid peroxidation were determined. Results: Acrylamide could induce premature senescence evident by the elevated lipid peroxidation and SA-β-gal activity and declined cell viability and glutathione. Moreover, carvacrol showed beneficial effects on both acrylamide- and H2O2-induced cellular senescence by significantly reversing or subsiding the effect of oxidative stress and mediating its consequences. Conclusions: It can be concluded that carvacrol has protective effects against oxidative cellular senescence induced by acrylamide in the NIH 3T3 cell line.

Short antimicrobial peptidomimetic SAMP-5 effective against multidrug-resistant gram-negative bacteria

SAMP-5 is a short histidine-derived antimicrobial peptidomimetic with pendant dialkylated tail. In this study, we evaluated the potential of SAMP-5 as an antimicrobial agent to combat multidrug-resistant gram-negative bacteria. SAMP-5 showed potent antimicrobial activity (minimum inhibitory concentration 16-64 μg/ml) comparable to melittin against multidrug-resistant Escherichia coli (MDREC) and multidrug-resistant (MDRPA). SAMP-5 displayed no cytotoxicity against three mammalian cells such as mouse macrophage RAW264.7, mouse embryonic fibroblast NIH-3T3, and human bone marrow SH-SY5Y cells at the concentration of 128 μg/ml. SAMP-5 showed resistance to proteolytic degradation with pepsin, trypsin, α-chymotrypsin, and proteinase K. Importantly, unlike ciprofloxacin, no antibiotic resistance against SAMP-5 arose for Pseudomonas aeruginosa during 7 days of serial passage at 0.5 × MIC. Moreover, SAMP-5 showed synergy or additive effects against MDRPA and MDREC, when it combined with chloramphenicol, ciprofloxacin, and oxacillin. Collectively, our results suggested that SAMP-5 is a promising alternative and adjuvant to treat infections caused by multidrug-resistant gram-negative bacteria.

Ufbp1, a Key Player of Ufm1 Conjugation System, Protects Against Ketosis-Induced Liver Injury via Suppressing Smad3 Activation

The dairy cattle suffer from severe liver dysfunction during the pathogenesis of ketosis. The Ufm1 conjugation system is crucial for liver development and homeostasis. Ufm1 binding protein (Ufbp1) is a putative Ufm1 target and an integral component, but its role in ketosis-induced liver injury is unclear so far. The purpose of this study is to explore the key role of Ufbp1 in liver fibrosis caused by ketosis in vivo and in vitro. Liver tissues were collected from ketotic cows and Ufbp1 conditional knockout (CKO) mice in vivo. However, Ufbp1–/– mouse embryonic fibroblast cells and Hela cells were used for in vitro validation. Subsequently, various assays were performed to reveal the underlying molecular mechanisms of the Ufbp1 protective effect. In this study, hepatic fibrosis, endoplasmic reticulum (ER) stress, and apoptosis were reported in the liver of ketotic cows, fibrotic markers (alpha-smooth muscle actin, Collagen1) and ER stress markers (glucose-regulated protein 78, CEBP homologous protein) were upregulated remarkably, and the apoptosis-related genes (Bcl2, Bax) were in line with expectations. Interestingly, Ufbp1 expression was almost disappeared, and Smad2/Smad3 protein was largely phosphorylated in the liver of ketotic cows, but Ufbp1 deletion caused Smad3 phosphorylation apparently, rather than Smad2, and elevated ER stress was observed in the CKO mice model. At the cellular level, Ufbp1 deficiency led to serious fibrotic and ER stress response, Smad3 was activated by phosphorylation significantly and then was translocated into the nucleus, whereas p-Smad2 was largely unaffected in embryonic fibroblast cells. Ufbp1 overexpression obviously suppressed Smad3 phosphorylation in Hela cells. Ufbp1 was found to be in full combination with Smad3 using endogenous immunoprecipitation. Taken together, our findings suggest that downregulation or ablation of Ufbp1 leads to Smad3 activation, elevated ER stress, and hepatocyte apoptosis, which in turn causes liver fibrosis. Ufbp1 plays a protective role in ketosis-induced liver injury.

Vimentin Cytoskeleton Architecture Analysis on Polylactide and Polyhydroxyoctanoate Substrates for Cell Culturing

Polylactide (PLA), widely used in bioengineering and medicine, gained popularity due to its biocompatibility and biodegradability. Natural origin and eco-friendly background encourage the search of novel materials with such features, such as polyhydroxyoctanoate (P(3HO)), a polyester of bacterial origin. Physicochemical features of both P(3HO) and PLA have an impact on cellular response 32, i.e., adhesion, migration, and cell morphology, based on the signaling and changes in the architecture of the three cytoskeletal networks: microfilaments (F-actin), microtubules, and intermediate filaments (IF). To investigate the role of IF in the cellular response to the substrate, we focused on vimentin intermediate filaments (VIFs), present in mouse embryonic fibroblast cells (MEF). VIFs maintain cell integrity and protect it from external mechanical stress, and also take part in the transmission of signals from the exterior of the cell to its inner organelles, which is under constant investigation. Physiochemical properties of a substrate have an impact on cells’ morphology, and thus on cytoskeleton network signaling and assembly. In this work, we show how PLA and P(3HO) crystallinity and hydrophilicity influence VIFs, and we identify that two different types of vimentin cytoskeleton architecture: network “classic” and “nutshell-like” are expressed by MEFs in different numbers of cells depending on substrate features.