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.
To identify long-term trajectories of physical function and health-related quality of life (HRQoL) among people with hip and/or knee osteoarthritis (HKOA) and the sociodemographic, lifestyle, and ...clinical factors associated with different trajectories.
Participants with HKOA from the EpiDoC study, a 10-year follow-up (2011-2021) population-based cohort, were considered. Sociodemographic, lifestyle, and clinical variables were collected at baseline in a structured interview and clinical appointment. Physical function and HRQoL were evaluated with the Health Assessment Questionnaire (HAQ) and EuroQoL, respectively, at baseline and the three follow-ups. Group-based trajectory modeling identified physical function and HRQoL trajectories. Multinomial logistic regression analyzed the associations between the covariates of interest and trajectory assignment (p < 0.05).
We included 983 participants with HKOA. We identified three trajectories for each outcome: "consistently low disability" (32.0%), "slightly worsening moderate disability" (47.0%), and "consistently high disability" (21.0%) for physical function; "consistently high HRQoL" (18.3%), "consistently moderate HRQoL" (48.4%) and "consistently low HRQoL" (33.4%) for HRQoL. Age ≥ 75 years, female sex, multimorbidity, and high baseline clinical severity were associated with higher risk of assignment to poorer physical function and HRQoL trajectories. Participants with high education level and with regular physical activity had a lower risk of assignment to a poor trajectory. Unmanageable pain levels increased the risk of assignment to the "consistently moderate HRQoL" trajectory.
Although the trajectories of physical function and HRQoL remained stable over 10 years, approximately 70% of people with HKOA maintained moderate or low physical function and HRQoL over this period. Modifiable risk factors like physical activity, multimorbidity and clinical severity were associated with poorer physical function and HRQoL trajectories. These risk factors may be considered in tailored healthcare interventions.
Alzheimer's disease (AD) is the most common neurodegenerative disorder, neuropathologically characterized by aggregates of β-amyloid peptides, which deposit as senile plaques, and of TAU protein, ...which forms neurofibrillary tangles. It is now widely accepted that neuroinflammation is implicated in AD pathogenesis.
Indeed, inflammatory mediators, such as cytokines and chemokines (chemotactic cytokines) can impact on the Alzheimer´s amyloid precursor protein by affecting its expression levels and amyloidogenic processing and/or β -amyloid aggregation. Additionally, cytokines and chemokines can influence kinases' activities, leading to abnormal TAU phosphorylation. To date there is no cure for AD, but several therapeutic strategies have been directed to prevent neuroinflammation. Anti-inflammatory, but also anti-amyloidogenic compounds, such as flavonoids were shown to favourably modulate some pathological events associated with neurodegeneration.
This review focuses on the role of cytokines and chemokines in AD-associated pathologies, and summarizes the potential anti-inflammatory therapeutic approaches aimed at preventing or slowing down disease progression.
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.
•Microtubule-independent antimitotics have emerged as new mitotic inhibitors, but failed in monotherapy clinical trials.•Inhibition of anti-apoptosis modulators in combination with SAC modifiers is a promising approach.•Strategies to circumvent mitotic slippage in order to increase susceptibility of cancer cells to death are reviewed.
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.
The initiation of an intestinal tumour is a probabilistic process that depends on the competition between mutant and normal epithelial stem cells in crypts
. Intestinal stem cells are closely ...associated with a diverse but poorly characterized network of mesenchymal cell types
. However, whether the physiological mesenchymal microenvironment of mutant stem cells affects tumour initiation remains unknown. Here we provide in vivo evidence that the mesenchymal niche controls tumour initiation in trans. By characterizing the heterogeneity of the intestinal mesenchyme using single-cell RNA-sequencing analysis, we identified a population of rare pericryptal Ptgs2-expressing fibroblasts that constitutively process arachidonic acid into highly labile prostaglandin E
(PGE
). Specific ablation of Ptgs2 in fibroblasts was sufficient to prevent tumour initiation in two different models of sporadic, autochthonous tumorigenesis. Mechanistically, single-cell RNA-sequencing analyses of a mesenchymal niche model showed that fibroblast-derived PGE
drives the expansion οf a population of Sca-1
reserve-like stem cells. These express a strong regenerative/tumorigenic program, driven by the Hippo pathway effector Yap. In vivo, Yap is indispensable for Sca-1
cell expansion and early tumour initiation and displays a nuclear localization in both mouse and human adenomas. Using organoid experiments, we identified a molecular mechanism whereby PGE
promotes Yap dephosphorylation, nuclear translocation and transcriptional activity by signalling through the receptor Ptger4. Epithelial-specific ablation of Ptger4 misdirected the regenerative reprogramming of stem cells and prevented Sca-1
cell expansion and sporadic tumour initiation in mutant mice, thereby demonstrating the robust paracrine control of tumour-initiating stem cells by PGE
-Ptger4. Analyses of patient-derived organoids established that PGE
-PTGER4 also regulates stem-cell function in humans. Our study demonstrates that initiation of colorectal cancer is orchestrated by the mesenchymal niche and reveals a mechanism by which rare pericryptal Ptgs2-expressing fibroblasts exert paracrine control over tumour-initiating stem cells via the druggable PGE
-Ptger4-Yap signalling axis.
Staphylococcus epidermidis biofilm-related infections are a current concern within the medical community due to their high incidence and prevalence, particularly in patients with indwelling medical ...devices. Biofilm gene expression analysis by quantitative real-time PCR (qPCR) has been increasingly used to understand the role of biofilm formation in the pathogenesis of S. epidermidis infections. However, depending on the RNA extraction procedure, and cDNA synthesis and qPCR master mixes used, gene expression quantification can be suboptimal. We recently showed that some RNA extraction kits are not suitable for S. epidermidis biofilms, due to sample composition, in particular the presence of the extracellular matrix. In this work, we describe a custom RNA extraction assay followed by the evaluation of gene expression using different commercial reverse transcriptase kits and qPCR master mixes. Our custom RNA extraction assay was able to produce good quality RNA with reproducible gene expression quantification, reducing the time and the costs associated. We also tested the effect of reducing cDNA and qPCR reaction volumes and, in most of the cases tested, no significant differences were found. Finally, we titered the SYBR Green I concentrations in standard PCR master mixes and compared the normalized expression of the genes icaA, bhp, aap, psmβ1 and agrB using 4 distinct biofilm forming S. epidermidis strains to the results obtained with commercially available kits. The overall results demonstrated that despite some statistically, but not biologically significant differences observed, the customized qPCR protocol resulted in the same gene expression trend presented by the commercially available kits used.
Nosocomial pneumonia has correlated to dental plaque and to oropharynx colonization in patients receiving mechanical ventilation. The interruption of this process, by preventing colonization of ...pathogenic bacteria, represents a potential procedure for the prevention of ventilator-associated pneumonia (VAP).
The study design was a prospective, randomized trial to verify if oral hygiene through toothbrushing plus chlorhexidine in gel at 0.12% reduces the incidence of ventilatior-associated pneumonia, the duration of mechanical ventilation, the length of hospital stay and the mortality rate in ICUs, when compared to oral hygiene only with chlorhexidine, solution of 0.12%, without toothbrushing, in adult individuals under mechanical ventilation, hospitalized in Clinical/Surgical and Cardiology Intensive Care Units (ICU). The study protocol was approved by the Ethical Committee of Research of the Health Sciences Center of the Federal University of Pernambuco - Certificate of Ethical Committee Approval (CAAE) 04300012500005208. Because it was a randomized trial, the research used CONSORT 2010 checklist criteria.
Seven hundred sixteen patients were admitted into the ICU; 219 fulfilled the criteria for inclusion and 213 patients were included; 108 were randomized to control group and 105 to intervention group. Toothbrushing plus 0.12% chlorhexidine gel demonstrated a lower incidence of VAP throughout the follow up period, although the difference was not statistically significant (p = 0.084). There was a significant reduction of the mean time of mechanical ventilation in the toothbrushing group (p = 0.018). Regarding the length of hospital stay in the ICU and mortality rates, the difference was not statistically significant (p = 0.064).
The results obtained showed that, among patients undergoing toothbrushing there was a significant reduction in duration of mechanical ventilation, and a tendency to reduce the incidence of VAP and length of ICU stay, although without statistical significance.
Retrospectively registered in the Brazilian Clinical Trials Registry (Registro Brasileiro de Ensaios Clínicos) - RBR-4TWH4M (4 September 2016).
Chronic wounds afford a hostile environment of damaged tissues that allow bacterial proliferation and further wound colonization.
is among the most common colonizers of infected wounds and it is a ...prolific biofilm former. Living in biofilm communities, cells are protected, become more difficult to control and eradicate, and less susceptible to antibiotic therapy. This work presents insights into the proceedings triggering
biofilm control with phage, honey, and their combination, achieved through standard antimicrobial activity assays, zeta potential and flow cytometry studies and further visual insights sought by scanning electron microscopy and transmission electron microscopy. Two Portuguese honeys (PF2 and U3) with different floral origin and an
-specific phage (EC3a), possessing depolymerase activity, were tested against 24- and 48-h-old biofilms. Synergic and additive effects were perceived in some phage-honey experiments. Combined therapy prompted similar phenomena in biofilm cells, visualized by electron microscopy, as the individual treatments. Honey caused minor membrane perturbations to complete collapse and consequent discharge of cytoplasmic content, and phage completely destroyed cells leaving only vesicle-like structures and debris. Our experiments show that the addition of phage to low honey concentrations is advantageous, and that even fourfold diluted honey combined with phage, presents no loss of antibacterial activity toward
. Portuguese honeys possess excellent antibiofilm activity and may be potential alternative therapeutic agents in biofilm-related wound infection. Furthermore, to our knowledge this is the first study that assessed the impacts of phage-honey combinations in bacterial cells. The synergistic effect obtained was shown to be promising, since the antiviral effect of honey limits the emergence of phage resistant phenotypes.
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