Filamin A-Hinge Region 1-EGFP: A Novel Tool for Tracking the Cellular Functions of Filamin A in Real-Time

Background: Filamin A (FLNa) is an actin-crosslinking protein necessary for stabilizing the cell surface, organizing protrusive activity and for promoting efficient cellular translocation. Recently, our group demonstrated the requirement of FLNa for the internalization of the chemokine receptor CCR2B. Methodology and Principal Findings: In order to study the role of FLNa in vitro and in real-time, we have developed a fluorescent FLNa-EGFP construct. In this novel imaging tool, we introduced the EGFP-tag inside the flexible hinge 1 region of FLNa between two calpain cleavage sites. Our findings indicate that the FLNa-EGFP construct was correctly expressed, cleaved by calpain and colocalized with actin filaments as shown by immunostaining experiments in the human melanoma cell lines A7 (FLNa-repleted) and M2 (FLNa-deficient). In addition, scanning-electron microscopy (SEM) and micropatterning studies also provided clear evidence that the cell rigidity was restored. FLNa-EGFP allowed us to demonstrate the interaction of FLNa with the chemokine receptor CCR2B in endocytic vesicles after CCL2 ligand stimulation. Through live-cell imaging studies we show that the CCR2B receptor in Rab5-positive vesicles moves along filamin A-positive fibers. Significance: Taken together, these results outline the functionality of the FLNa-EGFP and the importance of filamin A for receptor internalization and movement into endocytic vesicles

Biomarkers common for inflammatory periodontal disease and depression: A systematic review

Background Dysregulated immune response arising in the periphery can induce depressive symptoms through neuroimmune interactions. Inflammatory oral pathology can be a potent inducer of chronic neuroimmune response relevant to depression. We aimed to synthesize available evidence for the association between inflammatory periodontal diseases (IPD) and major depression (MD) in relation to a broad range of biomarkers. Methods Medline, Embase, PsycInfo, Cochrane Library, Web of Science and Scopus databases were searched from inception until January 27, 2022. Search terms included subject headings and synonyms for inflammatory periodontal disease and depression. Studies that reported data on both depression and inflammatory periodontal disease as categories along with measurement of a biomarker were considered. Two reviewers independently selected the articles for inclusion, extracted data and assessed the quality of each study. The protocol for this study was registered with PROSPERO, CRD42021215524. Results Twenty-eight studies were included in the final review-eleven cross-sectional studies, seven case-control studies, and six prospective cohort studies conducted in humans; the remaining four were experimental animal studies. Eighteen studies including all animal studies reported a positive association between depression and periodontal disease; one study reported a negative association and another nine studies found no such associations. Twenty studies reported mixed associations between IPD and biomarkers (i.e, salivary, serum, urine or gingival crevicular fluid cortisol, C reactive protein, cytokines, etc.). Biomarkers related to depression were gingival crevicular fluid cortisol, interleukin 6 (IL-6), Il-1β, immunoglobulin G against Bacterioides forsythus; root canal lipopolysaccharides; blood IL-6, IL-1β, cortisol, advanced oxidation protein products, nitric oxide metabolites, lipid hydroperoxides and trapping antioxidant parameter; whereas five studies found no associations between depression and a biomarker. Although animal studies showed interaction of immune, inflammatory and neurotrophic biomarkers in the relationship between depression and periodontal disease, human studies showed mixed findings. In most studies, there were risks of bias due to the sample selection and assessment protocol. Study heterogeneity and limited number of comparable studies reporting on shared biomarkers precluded a meta-analysis. Conclusion Immune-inflammatory contribution to depression was evident in the context of inflammatory periodontal diseases, but whether biomarkers mediate the associations between IPD and MD needs to be tested through methodologically rigorous studies aiming specifically at this hypothesis

Introduction of aromatic ring-containing substituents in cyclic nucleotides is associated with inhibition of toxin uptake by the hepatocyte transporters OATP 1B1 and 1B3

Analogs of the cyclic nucleotides cAMP and cGMP have been extensively used to mimic or modulate cellular events mediated by protein kinase A (PKA), Exchange protein directly activated by cAMP (Epac), or protein kinase G (PKG). We report here that some of the most commonly used cyclic nucleotide analogs inhibit transmembrane transport mediated by the liver specific organic anion transporter peptides OATP1B1 and OATP1B3, unrelated to actions on Epac, PKA or PKG. Several cAMP analogs, particularly with 8-pCPT-substitution, inhibited nodularin (Nod) induced primary rat hepatocyte apoptosis. Inhibition was not mediated by PKA or Epac, since increased endogenous cAMP, and some strong PKA- or Epacactivating analogs failed to protect cells against Nod induced apoptosis. The cAMP analogs inhibiting Nod induced hepatocyte apoptosis also reduced accumulation of radiolabeled Nod or cholic acid in primary rat hepatocytes. They also inhibited Nod induced apoptosis in HEK293 cells with enforced expression of OATP1B1 or 1B3, responsible for Nod transport into cells. Similar results were found with adenosine analogs, disconnecting the inhibitory effect of certain cAMP analogs from PKA or Epac. The most potent inhibitors were 8-pCPT-6-Phe-cAMP and 8-pCPT-29-O-Me-cAMP, whereas analogs like 6-MB-cAMP or 8-Br-cAMP did not inhibit Nod uptake. This suggests that the addition of aromatic ringcontaining substituents like the chloro-phenyl-thio group to the purines of cyclic nucleotides increases their ability to inhibit the OATP-mediated transport. Taken together, our data show that aromatic ring substituents can add unwanted effects to cyclic nucleotides, and that such nucleotide analogs must be used with care, particularly when working with cells expressing OATP1B1/1B3, like hepatocytes, or intact animals where hepatic metabolism can be an issue, as well as certain cancer cells. On the other hand, cAMP analogs with substituents like bromo, monobutyryl were non-inhibitory, and could be considered an alternative when working with cells expressing OATP1 family members

Iodinin (1,6-dihydroxyphenazine 5,10-dioxide) from streptosporangium sp. induces apoptosis selectively in myeloid leukemia cell lines and patient cells

-Despite recent improvement in therapy, acute myeloid leukemia (AML) is still associated with high lethality. In the presented study, we analyzed the bioactive compound iodinin (1,6-dihydroxyphenazine 5,10-dioxide) from a marine actinomycetes bacterium for the ability to induce cell death in a range of cell types. Iodinin showed selective toxicity to AML and acute promyelocytic (APL) leukemia cells, with EC50 values for cell death up to 40 times lower for leukemia cells when compared with normal cells. Iodinin also successfully induced cell death in patient-derived leukemia cells or cell lines with features associated with poor prognostic such as FLT3 internal tandem duplications or mutated/deficient p53. The cell death had typical apoptotic morphology, and activation of apoptotic signaling proteins like caspase-3. Molecular modeling suggested that iodinin could intercalate between bases in the DNA in a way similar to the anti-cancer drug daunorubicin (DNR), causing DNA-strand breaks. Iodinin induced apoptosis in several therapy-resistant AML-patient blasts, but to a low degree in peripheral blood leukocytes, and in contrast to DNR, not in rat cardiomyoblasts. The low activity towards normal cell types that are usually affected by anti-leukemia therapy suggests that iodinin and related compounds represent promising structures in the development of anti-cancer therapy.publishedVersio

Iodinin (1,6-dihydroxyphenazine 5,10-dioxide) from Streptosporangium sp. induces apoptosis selectively in myeloid leukemia cell lines and patient cells

Despite recent improvement in therapy, acute myeloid leukemia (AML) is still associated with high lethality. In the presented study, we analyzed the bioactive compound iodinin (1,6-dihydroxyphenazine 5,10-dioxide) from a marine actinomycetes bacterium for the ability to induce cell death in a range of cell types. Iodinin showed selective toxicity to AML and acute promyelocytic (APL) leukemia cells, with EC50 values for cell death up to 40 times lower for leukemia cells when compared with normal cells. Iodinin also successfully induced cell death in patient-derived leukemia cells or cell lines with features associated with poor prognostic such as FLT3 internal tandem duplications or mutated/deficient p53. The cell death had typical apoptotic morphology, and activation of apoptotic signaling proteins like caspase-3. Molecular modeling suggested that iodinin could intercalate between bases in the DNA in a way similar to the anti-cancer drug daunorubicin (DNR), causing DNA-strand breaks. Iodinin induced apoptosis in several therapy-resistant AML-patient blasts, but to a low degree in peripheral blood leukocytes, and in contrast to DNR, not in rat cardiomyoblasts. The low activity towards normal cell types that are usually affected by anti-leukemia therapy suggests that iodinin and related compounds represent promising structures in the development of anti-cancer therapy