A Pyranoxanthone as a Potent Antimitotic and Sensitizer of Cancer Cells to Low Doses of Paclitaxel.

Microtubule-targeting agents (MTAs) remain a gold standard for the treatment of several cancer types. By interfering with microtubules dynamic, MTAs induce a mitotic arrest followed by cell death. This antimitotic activity of MTAs is dependent on the spindle assembly checkpoint (SAC), which monitors the integrity of the mitotic spindle and proper chromosome attachments to microtubules in order to ensure accurate chromosome segregation and timely anaphase onset. However, the cytotoxic activity of MTAs is restrained by drug resistance and/or toxicities, and had motivated the search for new compounds and/or alternative therapeutic strategies. Here, we describe the synthesis and mechanism of action of the xanthone derivative pyranoxanthone 2 that exhibits a potent anti-growth activity against cancer cells. We found that cancer cells treated with the pyranoxanthone 2 exhibited persistent defects in chromosome congression during mitosis that were not corrected over time, which induced a prolonged SAC-dependent mitotic arrest followed by massive apoptosis. Importantly, pyranoxanthone 2 was able to potentiate apoptosis of cancer cells treated with nanomolar concentrations of paclitaxel. Our data identified the potential of the pyranoxanthone 2 as a new potent antimitotic with promising antitumor potential, either alone or in combination regimens.

Eosinophilic Variant of Granulomatosis With Polyangiitis.

Granulomatosis with polyangiitis (GPA) is a multisystemic necrotizing vasculitis with a special tropism to the respiratory tract and the kidneys. Although uncommon, GPA may be associated with hypereosinophilia and limited organ involvement. In these cases, American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) classification criteria may be insufficient to establish the diagnosis. We described a limited form of GPA, hypereosinophilia, and predominant skin involvement.

Undiagnosed Primary Sjögren's Syndrome With Pleural Involvement.

Sjögren's syndrome (SS) is a systemic autoimmune disorder characterized by the lymphocytic infiltration of the exocrine glands, primarily the salivary and lacrimal glands, resulting in the dryness of the eyes and mouth. However, Sjögren's syndrome is not limited to glandular involvement, as it can also affect various other organ systems, leading to a wide range of extraglandular manifestations and delaying the diagnosis. In this scenario, a high level of suspicion is required.

SARS-CoV-2 infection induces DNA damage, through CHK1 degradation and impaired 53BP1 recruitment, and cellular senescence.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the RNA virus responsible for the coronavirus disease 2019 (COVID-19) pandemic. Although SARS-CoV-2 was reported to alter several cellular pathways, its impact on DNA integrity and the mechanisms involved remain unknown. Here we show that SARS-CoV-2 causes DNA damage and elicits an altered DNA damage response. Mechanistically, SARS-CoV-2 proteins ORF6 and NSP13 cause degradation of the DNA damage response kinase CHK1 through proteasome and autophagy, respectively. CHK1 loss leads to deoxynucleoside triphosphate (dNTP) shortage, causing impaired S-phase progression, DNA damage, pro-inflammatory pathways activation and cellular senescence. Supplementation of deoxynucleosides reduces that. Furthermore, SARS-CoV-2 N-protein impairs 53BP1 focal recruitment by interfering with damage-induced long non-coding RNAs, thus reducing DNA repair. Key observations are recapitulated in SARS-CoV-2-infected mice and patients with COVID-19. We propose that SARS-CoV-2, by boosting ribonucleoside triphosphate levels to promote its replication at the expense of dNTPs and by hijacking damage-induced long non-coding RNAs' biology, threatens genome integrity and causes altered DNA damage response activation, induction of inflammation and cellular senescence.

Navitoclax Enhances the Therapeutic Effects of PLK1 Targeting on Lung Cancer Cells in 2D and 3D Culture Systems.

The efficacy of antimitotics is limited by slippage, whereby treated cells arrested in mitosis exit mitosis without cell division and, eventually, escape apoptosis, constituting a serious resistance mechanism to antimitotics. Strategies to overcome slippage should potentiate the cancer cell killing activity of these antimitotics. Such strategies should accelerate cell death in mitosis before slippage. Here, we undertook a mechanistic analysis to test whether the apoptosis activator Navitoclax potentiates apoptosis triggered by the antimitotic BI2536, a potent inhibitor of Polo-like kinase 1 (PLK1) with the goal of overcoming slippage. We found that cancer cells in 2D cultures treated with BI2536 alone accumulate in mitosis, but a significant fraction of arrested cells undergo slippage and survive. Remarkably, combining BI2536 with Navitoclax dramatically reduces slippage, shifting the cell fate to accelerated death in mitosis. The results are confirmed in 3D spheroids, a preclinical system that mimics in vivo tumor drug responses. Importantly, in 3D spheroids, the effect of the BI2536/Navitoclax combination requires a lower therapeutic dosage of each drug, underlying its potential to improve the therapeutic index. Our results highlight the relevance of apoptosis potentiators to circumvent slippage associated with antimitotics. The combination of BI2536 with Navitoclax shows in vitro synergy/additive effect, which warrants further clinical research.

Antagonizing the spindle assembly checkpoint silencing enhances paclitaxel and Navitoclax-mediated apoptosis with distinct mechanistic.

Antimitotic drugs arrest cells in mitosis through chronic activation of the spindle assembly checkpoint (SAC), leading to cell death. However, drug-treated cancer cells can escape death by undergoing mitotic slippage, due to premature mitotic exit. Therefore, overcoming slippage issue is a promising chemotherapeutic strategy to improve the effectiveness of antimitotics. Here, we antagonized SAC silencing by knocking down the MAD2-binding protein p31comet, to delay mitotic slippage, and tracked cancer cells treated with the antimitotic drug paclitaxel, over 3 days live-cell time-lapse analysis. We found that in the absence of p31comet, the duration of mitotic block was increased in cells challenged with nanomolar concentrations of paclitaxel, leading to an additive effects in terms of cell death which was predominantly anticipated during the first mitosis. As accumulation of an apoptotic signal was suggested to prevent mitotic slippage, when we challenged p31comet-depleted mitotic-arrested cells with the apoptosis potentiator Navitoclax (previously called ABT-263), cell fate was shifted to accelerated post-mitotic death. We conclude that inhibition of SAC silencing is critical for enhancing the lethality of antimitotic drugs as well as that of therapeutic apoptosis-inducing small molecules, with distinct mechanisms. The study highlights the potential of p31comet as a target for antimitotic therapies.

The Mad2-Binding Protein p31comet as a Potential Target for Human Cancer Therapy.

The spindle assembly checkpoint (SAC) is a surveillance mechanism that prevents mitotic exit at the metaphase-to-anaphase transition until all chromosomes have established correct bipolar attachment to spindle microtubules. Activation of SAC relies on the assembly of the mitotic checkpoint complex (MCC), which requires conformational change from inactive open Mad2 (OMad2) to the active closed Mad2 (C-Mad2) at unattached kinetochores. The Mad2-binding protein p31comet plays a key role in controlling timely mitotic exit by promoting SAC silencing, through preventing Mad2 activation and promoting MCC disassembly. Besides, increasing evidences highlight the p31comet potential as target for cancer therapy. Here, we provide an updated overview of the functional significance of p31comet in mitotic progression, and discuss the potential of deregulated expression of p31comet in cancer and in therapeutic strategies.

Gut Microbiome Composition and Metabolic Status Are Differently Affected by Early Exposure to Unhealthy Diets in a Rat Model.

Childhood is a critical stage of development during which diet can have profound influence on the microbiota-host interactions, leading to potentially lifelong impacts. This study aimed to investigate whether the consumption of cafeteria diet (CAFD) and sugary drinks during early rat life alters the structure of the gut microbial community and the metabolic activity. Four-week-old male Wistar rats (n = 27) were fed a standard chow diet with ad libitum access to water (CD) or to sucrose solution (HSD), and a third group was fed with CAFD and a sucrose solution for 14 weeks. HSD and CAFD consumption induced alterations in Firmicutes to Bacteroidetes ratio, Proteobacteria, and Verrucomicrobia. HSD increased the abundance of Barnesiella, whereas CAFD induced a depletion of Saccharibacteria. CAFD increased total white adipose tissue (WAT) weight (p < 0.0005) compared to CD. When CAFD was compared to HSD, a significant difference was found only for retroperitoneal WAT (p < 0.0005). Unhealthy diet-fed groups presented higher glucose (p < 0.0005), total cholesterol and creatinine serum levels (p < 0.005) compared to the CD rats. Early-life consumption of HSD, and of CAFD even more so, can have long-lasting negative effects on metabolic function. The gut microbiota communities were distinctively perturbed by diet composition.

Three-Dimensional Spheroids as In Vitro Preclinical Models for Cancer Research.

Most cancer biologists still rely on conventional two-dimensional (2D) monolayer culture techniques to test in vitro anti-tumor drugs prior to in vivo testing. However, the vast majority of promising preclinical drugs have no or weak efficacy in real patients with tumors, thereby delaying the discovery of successful therapeutics. This is because 2D culture lacks cell-cell contacts and natural tumor microenvironment, important in tumor signaling and drug response, thereby resulting in a reduced malignant phenotype compared to the real tumor. In this sense, three-dimensional (3D) cultures of cancer cells that better recapitulate in vivo cell environments emerged as scientifically accurate and low cost cancer models for preclinical screening and testing of new drug candidates before moving to expensive and time-consuming animal models. Here, we provide a comprehensive overview of 3D tumor systems and highlight the strategies for spheroid construction and evaluation tools of targeted therapies, focusing on their applicability in cancer research. Examples of the applicability of 3D culture for the evaluation of the therapeutic efficacy of nanomedicines are discussed.

Mitosis inhibitors in anticancer therapy: When blocking the exit becomes a solution.

Current microtubule-targeting agents (MTAs) remain amongst the most important antimitotic drugs used against a broad range of malignancies. By perturbing spindle assembly, MTAs activate the spindle assembly checkpoint (SAC), which induces mitotic arrest and subsequent apoptosis. However, besides toxic side effects and resistance, mitotic slippage and failure in triggering apoptosis in various cancer cells are limiting factors of MTAs efficacy. Alternative strategies to target mitosis without affecting microtubules have, thus, led to the identification of small molecules, such as those that target spindle Kinesins, Aurora and Polo-like kinases. Unfortunately, these so-called second-generation of antimitotics, encompassing mitotic blockers and mitotic drivers, have failed in clinical trials. Our recent understanding regarding the mechanisms of cell death during a mitotic arrest pointed out apoptosis as the main variable, providing an opportunity to control the cell fates and influence the effectiveness of antimitotics. Here, we provide an overview on the second-generation of antimitotics, and discuss possible strategies that exploit SAC activity, mitotic slippage/exit and apoptosis induction, in order to improve the efficacy of anticancer strategies that target mitosis.

Chalcone derivatives targeting mitosis: synthesis, evaluation of antitumor activity and lipophilicity.

This study describes the synthesis of a series of chalcones, including pyrazole and α,ß-epoxide derivatives, and evaluation of their cell growth inhibitory activity in three human tumor cell lines, as well as their lipophilicity using liposomes as a biomimetic membrane model. Structure-activity and structure-lipophilicity relationships were established for the synthetized chalcones. From this work, nine chalcones (3, 5, 9, 11, 15-19) showing suitable drug-like lipophilicity with potent growth inhibitory activity were identified, being the growth inhibitory effect of compounds 15-17 associated with a pronounced antimitotic effect. Compounds 15-17 affected spindle assembly and, as a consequence, arrested cells at metaphase in NCI-H460 cells, culminating in cell death. Amongst the compounds tested, compound 15 exhibited the highest antimitotic activity as revealed by mitotic index calculation. Moreover, 15 was able to enhance chemosensitivity of tumor cells to low doses of paclitaxel in NCI-H460 cells. The results indicate that 15 exerts its antiproliferative activity by affecting microtubules and causing cell death subsequently to a mitotic arrest, and thus has the potential for antitumor activity.

Simple Protocol for Molecular Fingerprinting of Human Oral Microbiota Samples in Lab Classes.

DNA fingerprinting is a major tool in identifying individuals and in evidence matching. However, this technique can be difficult to reproduce in practical classes. Here, we report on distinct PCR profiles obtained when amplifying saliva DNA of a score of distinct individuals with Random Amplified Polymorphic DNA (RAPD)-PCR primer BOXA1R. The RAPD-PCR method is simple and efficient for discrimination between bacterial strains and is used in this instance to obtain personalized fingerprints of each individual's oral microbiota. We present real results with undergraduate students confirming that this procedure is easily feasible in practical classes. Based on the results presented, we suggest a laboratory activity for undergraduate Molecular Biology or Microbiology students.

Metagenomic survey of methanesulfonic acid (MSA) catabolic genes in an Atlantic Ocean surface water sample and in a partial enrichment.

Methanesulfonic acid (MSA) is a relevant intermediate of the biogeochemical cycle of sulfur and environmental microorganisms assume an important role in the mineralization of this compound. Several methylotrophic bacterial strains able to grow on MSA have been isolated from soil or marine water and two conserved operons, msmABCD coding for MSA monooxygenase and msmEFGH coding for a transport system, have been repeatedly encountered in most of these strains. Homologous sequences have also been amplified directly from the environment or observed in marine metagenomic data, but these showed a base composition (G + C content) very different from their counterparts from cultivated bacteria. The aim of this study was to understand which microorganisms within the coastal surface oceanic microflora responded to MSA as a nutrient and how the community evolved in the early phases of an enrichment by means of metagenome and gene-targeted amplicon sequencing. From the phylogenetic point of view, the community shifted significantly with the disappearance of all signals related to the Archaea, the Pelagibacteraceae and phylum SAR406, and the increase in methylotroph-harboring taxa, accompanied by other groups so far not known to comprise methylotrophs such as the Hyphomonadaceae. At the functional level, the abundance of several genes related to sulfur metabolism and methylotrophy increased during the enrichment and the allelic distribution of gene msmA diagnostic for MSA monooxygenase altered considerably. Even more dramatic was the disappearance of MSA import-related gene msmE, which suggests that alternative transporters must be present in the enriched community and illustrate the inadequacy of msmE as an ecofunctional marker for MSA degradation at sea.

Genome Sequence of Rhodococcus sp. Strain RD6.2 DSM 46800, a Methanesulfonate-Degrading Strain.

The complete genome sequence of a methanesulfonate-degrading strain, Rhodococcus sp. strain RD6.2 DSM 46800, which was isolated from a brackish marsh sediment sample, is described here. This is the first reported genome of a nonproteobacterial strain using methanesulfonate (MSA) as a sole source of carbon and energy, which does not possess the conventional MSA-monooxygenase (MSAMO).

Complete Genome Sequences of Two Strains of "Candidatus Filomicrobium marinum," a Methanesulfonate-Degrading Species.

Two novel methanesulfonate-degrading bacterial strains of "Candidatus Filomicrobium marinum" (strains Y and W) were isolated from a marine water enrichment, and their complete genome sequences are presented here. These are the first full genomes reported for the genus Filomicrobium and for methanesulfonate (MSA)-degrading bacteria.

Methanesulfonate (MSA) Catabolic Genes from Marine and Estuarine Bacteria.

Quantitatively, methanesulfonate (MSA) is a very relevant compound in the global biogeochemical sulfur cycle. Its utilization by bacteria as a source of carbon and energy has been described and a specific enzyme, methanesulfonate monooxygenase (MSAMO), has been found to perform the first catabolic step of its oxidation. Other proteins seemingly involved in the import of MSA into bacterial cells have been reported. In this study, we obtained novel sequences of genes msmA and msmE from marine, estuary and soil MSA-degraders (encoding the large subunit of the MSAMO enzyme and the periplasmic component of the import system, respectively). We also obtained whole-genome sequences of two novel marine Filomicrobium strains, Y and W, and annotated two full msm operons in these genomes. Furthermore, msmA and msmE sequences were amplified from North Atlantic seawater and analyzed. Good conservation of the MsmA deduced protein sequence was observed in both cultured strains and metagenomic clones. A long spacer sequence in the Rieske-type [2Fe-2S] cluster-binding motif within MsmA was found to be conserved in all instances, supporting the hypothesis that this feature is specific to the large (α) subunit of the MSAMO enzyme. The msmE gene was more difficult to amplify, from both cultivated isolates and marine metagenomic DNA. However, 3 novel msmE sequences were obtained from isolated strains and one directly from seawater. With both genes, our results combined with previous metagenomic analyses seem to imply that moderate to high-GC strains are somehow favored during enrichment and isolation of MSA-utilizing bacteria, while the majority of msm genes obtained by cultivation-independent methods have low levels of GC%, which is a clear example of the misrepresentation of natural populations that culturing, more often than not, entails. Nevertheless, the data obtained in this work show that MSA-degrading bacteria are abundant in surface seawater, which suggests ecological relevance for this metabolic group of bacteria.

Endothelial dysfunction after pregnancy-induced hypertension.

OBJECTIVE: To carry out long-term analysis of the presence of endothelial dysfunction after the development of pregnancy-induced hypertension (PIH). METHODS: In a retrospective cohort study, data were analyzed from 60 women who delivered at a tertiary maternity hospital in Fortaleza, Ceara, Brazil, between 1992 and 2002. Thirty women had a history of PIH and 30 had no history of complications. Anthropometric and laboratory data were collected, and endothelial function was evaluated by flow-mediated dilatation of the brachial artery. Continuous variables were analyzed via Student t test, and Mann-Whitney test was used to compare means. Clinical and metabolic measures were categorized according to cardiovascular risk by cutoff points determined by national consensus; χ2 and Fisher exact tests were used to compare the groups. Relative risk was calculated for variables that were statistically significant (P<0.05). RESULTS: Women with a history of PIH had higher body mass index (P=0.03), systolic blood pressure (P=0.03), low-density lipoprotein cholesterol (P=0.02), and fasting glucose (P=0.02) compared with women with no pregnancy complications. The frequency of endothelial dysfunction was 60% among all women, with a significant difference between the 2 groups (P=0.01). CONCLUSION: Women with a history of PIH were found to have a higher frequency of long-term endothelial dysfunction.