Defects of mitochondria-lysosomes communication induce secretion of mitochondria-derived vesicles and drive chemoresistance in ovarian cancer cells.

BACKGROUND: Among the mechanisms of mitochondrial quality control (MQC), generation of mitochondria-derived vesicles (MDVs) is a process to avoid complete failure of mitochondria determining lysosomal degradation of mitochondrial damaged proteins. In this context, RAB7, a late endocytic small GTPase, controls delivery of MDVs to late endosomes for subsequent lysosomal degradation. We previously demonstrated that RAB7 has a pivotal role in response to cisplatin (CDDP) regulating resistance to the drug by extracellular vesicle (EVs) secretion. METHODS: Western blot and immunofluorescence analysis were used to analyze structure and function of endosomes and lysosomes in CDDP chemosensitive and chemoresistant ovarian cancer cell lines. EVs were purified from chemosensitive and chemoresistant cells by ultracentrifugation or immunoisolation to analyze their mitochondrial DNA and protein content. Treatment with cyanide m-chlorophenylhydrazone (CCCP) and RAB7 modulation were used, respectively, to understand the role of mitochondrial and late endosomal/lysosomal alterations on MDV secretion. Using conditioned media from chemoresistant cells the effect of MDVs on the viability after CDDP treatment was determined. Seahorse assays and immunofluorescence analysis were used to study the biochemical role of MDVs and the uptake and intracellular localization of MDVs, respectively. RESULTS: We observed that CDDP-chemoresistant cells are characterized by increased MDV secretion, impairment of late endocytic traffic, RAB7 downregulation, an increase of RAB7 in EVs, compared to chemosensitive cells, and downregulation of the TFEB-mTOR pathway overseeing lysosomal and mitochondrial biogenesis and turnover. We established that MDVs can be secreted rather than delivered to lysosomes and are able to deliver CDDP outside the cells. We showed increased secretion of MDVs by chemoresistant cells ultimately caused by the extrusion of RAB7 in EVs, resulting in a dramatic drop in its intracellular content, as a novel mechanism to regulate RAB7 levels. We demonstrated that MDVs purified from chemoresistant cells induce chemoresistance in RAB7-modulated process, and, after uptake from recipient cells, MDVs localize to mitochondria and slow down mitochondrial activity. CONCLUSIONS: Dysfunctional MQC in chemoresistant cells determines a block in lysosomal degradation of MDVs and their consequent secretion, suggesting that MQC is not able to eliminate damaged mitochondria whose components are secreted becoming effectors and potential markers of chemoresistance.

Risk management and communication plans from SARS to COVID-19 and beyond.

OBJECTIVE: Nowadays, due to globalisation, the likelihood that infectious diseases spread rapidly is extraordinarily high. SARS and COVID-19 are two diseases of the Coronavirus family, which developed in China and then spread internationally, causing global public health emergencies. This study investigates the role that risk management and communication systems played in mitigating these emergencies, to establish how they should be improved in the future. METHODS: A narrative review was carried out to investigate different knowledge domains, such as risk management and communication, risk assessment and indicators, epidemiological and clinical data, diagnostic methods, vaccines, public health and social measures. RESULTS: On one side, risk management systems assess the main data, knowledge, and indicators on epidemiology, diagnostics, and vaccines (science-based); on the other side, they apply public health and social measures (socially-based). Decision-makers, in fact, implement their actions by constantly balancing these two sides (policy-based). CONCLUSIONS: A correct crisis management approach should support the governance of pandemics, by harmonising the actual risks assessed by experts with those perceived by the general population. It should incorporate not only the biological, but even the environmental, social and economic aspects of virus emergencies, towards establishing a suitable framework to deal with possible future pandemics.

Clinical features of COVID-19 and SARS epidemics. A literature review.

SARS-CoV-2, responsible for the current pandemic, is a novel strain of the Coronaviridae family, which has infected humans as a result of the leap to a new species. It causes an atypical pneumonia similar to that caused by SARS-CoV in 2003. SARS-CoV-2 has currently infected more than 9,200,000 people and caused almost 480,000 deaths worldwide. Although SARS-CoV-2 and SARS-CoV have similar phylogenetic and pathogenetic characteristics, they show important differences in clinical manifestations. We have reviewed the recent literature comparing the characteristics of the two epidemics and highlight their peculiar aspects. An analysis of all signs and symptoms of 3,365 SARS patients and 23,280 COVID-19 patients as well as of the comorbidities has been carried out. A total of 17 and 75 studies regarding patients with SARS and COVID-19, respectively, were included in the analysis. The analysis revealed an overlap of some symptoms between the two infections. Unlike SARS patients, COVID-19 patients have developed respiratory, neurological and gastrointestinal symptoms, and, in a limited number of subjects, symptoms involving organs such as skin and subcutaneous tissue, kidneys, cardiovascular system, liver and eyes. This analysis was conducted in order to direct towards an early identification of the infection, a suitable diagnostic procedure and the adoption of appropriate containment measures.

Tailoring of silica-based nanoporous pod by spermidine multi-activity.

Ubiquitous in nature, polyamines (PAs) are a class of low-molecular aliphatic amines critically involved in cell growth, survival and differentiation. The polycation behavior is validated as a successful strategy in delivery systems to enhance oligonucleotide loading and cellular uptake. In this study, the chemical features and the functional roles of the PA spermidine are synergistically exploited in the synthesis and bioactive functionalization of SiO2-based structures. Inspired by biosilicification, the role of spermidine is assessed both as catalyst and template in a biomimetic one-pot synthesis of dense silica-based particles (SPs) and as a competitive agent in an interfacial reassembly strategy, to empty out SPs and generate spermidine-decorated hollow silica nanoporous pods (spd-SNPs). Spermidine bioactivity is then employed for targeting tumor cell over-expressed polyamine transport system (PTS) and for effective delivery of functional miRNA into melanoma cells. Spermidine decoration promotes spd-SNP cell internalization mediated by PTS and along with hollow structure enhances oligonucleotide loading. Accordingly, the functional delivery of the tumor suppressor miR-34a 3p resulted in intracellular accumulation of histone-complexed DNA fragments associated with apoptosis. Overall, the results highlight the potential of spd-SNP as a multi-agent anticancer therapy.

Bio-Recognition in Spectroscopy-Based Biosensors for *Heavy Metals-Water and Waterborne Contamination Analysis.

: Microsystems and biomolecules integration as well multiplexing determinations are key aspects of sensing devices in the field of heavy metal contamination monitoring. The present review collects the most relevant information about optical biosensors development in the last decade. Focus is put on analytical characteristics and applications that are dependent on: (i) Signal transduction method (luminescence, colorimetry, evanescent wave (EW), surface-enhanced Raman spectroscopy (SERS), Förster resonance energy transfer (FRET), surface plasmon resonance (SPR)); (ii) biorecognition molecules employed (proteins, nucleic acids, aptamers, and enzymes). The biosensing systems applied (or applicable) to water and milk samples will be considered for a comparative analysis, with an emphasis on water as the primary source of possible contamination along the food chain.

Immune Profiling of Polysaccharide Submicron Vesicles.

Alginate (ALG) and chitosan (CS) have been extensively used for biomedical applications; however, data relative to immune responses exerted by them are scarce. We synthesized a submicron vesicle system (SV) displaying a CS shell over an ALG core. Intravenous injection of these promising carriers could be a possible route of delivery; therefore, we evaluated their impact on human peripheral blood mononuclear cells (PBMCs). By this ex vivo approach, we established how SV chemical-physical characteristics affected the immune cells in terms of cellular uptake, viability, and state of activation. By flow cytometry, we demonstrated that SVs were internalized by PBMCs with differential trends. No substantial necrotic and apoptotic signals were recorded, and SVs weakly affected activation status of PBMCs (concerning the markers CD69, CD25, CD80, and the cytokines TNF-α and IL-6), showing high immune biocompatibility and low immunomodulating properties. Our findings gain particular value toward the biomedical applications of SVs and make these polymer-based structures more attractive for translation into clinical uses.

Maghemite Nanoparticles with Enhanced Magnetic Properties: One-Pot Preparation and Ultrastable Dextran Shell.

In the field of nanomedicine, superparamagnetic nanoparticles are one of the most studied nanomaterials for theranostics. In this study, a one-pot synthesis of magnetic nanoparticles is presented, with an increased control on particle size from 10 to 40 nm. Monitoring of vacuum level is introduced here as a crucial parameter for achieving a fine particle morphology. The magnetic properties of these nanoparticles are highly affected by disorders or mismatches in crystal structure. A prolonged oxidation step is applied to the obtained nanoparticles to transform the magnetic phases into a pure maghemite one, confirmed by high-resolution X-ray photoelectron spectroscopy analysis, by Mössbauer spectrometry and, indirectly, by increased performances in magnetization curves and in relaxation times. Afterward, the attained nanoparticles are transferred into water by a nonderivatized dextran coating. Thermogravimetric analysis confirms that polysaccharide molecules replace oleic acid on the surface by stabilizing the particles in the aqueous phase and culture media. Preliminary in vitro test reveals that the dextran-coated nanoparticles are not passively internalized from the cells. As a proof of concept, a secondary layer of chitosan assures a positive charge to the nanoparticle surface, thus enhancing cellular internalization.

Radio Electric Asymmetric Conveyer Technology Modulates Neuroinflammation in a Mouse Model of Neurodegeneration.

In this study, the effects of Radio Electric Asymmetric Conveyer (REAC), a non-invasive physical treatment, on neuroinflammatory responses in a mouse model of parkinsonism induced by intoxication with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), were investigated in vivo. We found that the REAC tissue optimization treatment specific for neuro-regenerative purposes (REAC TO-RGN-N) attenuated the inflammatory picture evoked by MPTP-induced nigro-striatal damage in mice, decreasing the levels of pro-inflammatory molecules and increasing anti-inflammatory mediators. Besides, there was a significant reduction of both astrocyte and microglial activation in MPTP-treated mice exposed to REAC TO-RGN-N. These results indicated that REAC TO-RGN-N treatment modulates the pro-inflammatory responses and reduces neuronal damage in MPTP-induced parkinsonism.

Inhibin-A and Decorin Secreted by Human Adult Renal Stem/Progenitor Cells Through the TLR2 Engagement Induce Renal Tubular Cell Regeneration.

Acute kidney injury (AKI) is a public health problem worldwide. Several therapeutic strategies have been made to accelerate recovery and improve renal survival. Recent studies have shown that human adult renal progenitor cells (ARPCs) participate in kidney repair processes, and may be used as a possible treatment to promote regeneration in acute kidney injury. Here, we show that human tubular ARPCs (tARPCs) protect physically injured or chemically damaged renal proximal tubular epithelial cells (RPTECs) by preventing cisplatin-induced apoptosis and enhancing proliferation of survived cells. tARPCs without toll-like receptor 2 (TLR2) expression or TLR2 blocking completely abrogated this regenerative effect. Only tARPCs, and not glomerular ARPCs, were able to induce tubular cell regeneration process and it occurred only after damage detection. Moreover, we have found that ARPCs secreted inhibin-A and decorin following the RPTEC damage and that these secreted factors were directly involved in cell regeneration process. Polysaccharide synthetic vesicles containing these molecules were constructed and co-cultured with cisplatin damaged RPTECs. These synthetic vesicles were not only incorporated into the cells, but they were also able to induce a substantial increase in cell number and viability. The findings of this study increase the knowledge of renal repair processes and may be the first step in the development of new specific therapeutic strategies for renal repair.

Anti-aggregating effect of the naturally occurring dipeptide carnosine on aß1-42 fibril formation.

Carnosine is an endogenous dipeptide abundant in the central nervous system, where by acting as intracellular pH buffering molecule, Zn/Cu ion chelator, antioxidant and anti-crosslinking agent, it exerts a well-recognized multi-protective homeostatic function for neuronal and non-neuronal cells. Carnosine seems to counteract proteotoxicity and protein accumulation in neurodegenerative conditions, such as Alzheimer's Disease (AD). However, its direct impact on the dynamics of AD-related fibril formation remains uninvestigated. We considered the effects of carnosine on the formation of fibrils/aggregates of the amyloidogenic peptide fragment Aß1-42, a major hallmark of AD injury. Atomic force microscopy and thioflavin T assays showed inhibition of Aß1-42 fibrillogenesis in vitro and differences in the aggregation state of Aß1-42 small pre-fibrillar structures (monomers and small oligomers) in the presence of carnosine. in silico molecular docking supported the experimental data, calculating possible conformational carnosine/Aß1-42 interactions. Overall, our results suggest an effective role of carnosine against Aß1-42 aggregation.

Quartz crystal microbalance with dissipation (QCM-D) as tool to exploit antigen-antibody interactions in pancreatic ductal adenocarcinomadetection.

Novel synthetic peptides represent smart molecules for antigen-antibody interactions in several bioanalytics applications, from purification to serum screening. Their immobilization onto a solid phase is considered a key point for sensitivity increasing. In this view, we exploited Quartz Crystal Microbalance with simultaneous frequency and dissipation monitoring (QCM-D) with a double aim, specifically, as investigative tool for spacers monolayer assembling and its functional evaluation, as well as high sensitive method for specific immunosorbent assays. The method was applied to pancreatic ductal adenocarcinoma (PDAC) detection by studying the interactions between synthetic phosphorylated and un-phosphorylated α-enolase peptides with sera of healthy and PDAC patients. The synthetic peptides were immobilized on the gold surface of the QCM-D sensor via a self-assembled alkanethiol monolayer. The presented experimental results can be applied to the development of surfaces less sensitive to non-specific interactions with the final target to suggest specific protocols for detecting PDAC markers with un-labeled biosensors.

IRF5 promotes the proliferation of human thyroid cancer cells.

BACKGROUND: Interferon Regulatory Factor 5 is a transcription factor that regulates the expression of genes involved in the response to viral infection and in the stimulation of the immune system. Moreover, multiple studies have demonstrated that it negatively regulates cell growth and oncogenesis, favoring cell differentiation and apoptosis.Thyroid carcinoma represents 98% of all thyroid malignancies and has shown a steady increase in incidence in both the USA and western European countries. FINDINGS: We investigated the expression, localization and function of IRF5 in thyroid cancer cells and found that it is highly expressed in both primary and immortalized thyroid carcinomas but not in normal thyrocytes. IRF5 levels were variably modulated by Interferon alpha but IRF5 only localized in the cytoplasmic compartment, thus failing to induce p21 expression as previously reported in different cell models. Furthermore, ectopic IRF5 increased both the proliferation rate and the clonogenic potential of malignant thyroid cells, protecting them from the cytotoxic effects of DNA-damaging agents. These results were directly attributable to IRF5, as demonstrated by the reduction in colony-forming ability of thyroid cancer cells after IRF5 silencing. An IRF5-dependent induction of endogenous B-Raf observed in all thyroid cancer cells might contribute to these unexpected effects. CONCLUSIONS: These findings suggest that, in thyroid malignancies, IRF5 displays tumor-promoting rather than tumor-suppressor activities.

TEL/ARG induces cytoskeletal abnormalities in 293T cells.

We previously identified TEL/ARG as a novel fusion transcript consisting of the oligomerization domain of TEL and the kinase domain of ARG, in a case of acute myeloid leukemia. We report here the existence of an alternatively spliced TEL/ARG transcript lacking part of a F-actin binding domain of ARG, and the phenotype of TEL/ARG expressing 293T cells. In 293T cells, both TEL/ARG forms co-localized with the cellular beta-actin and were associated with a morphologic change of the cells, consisting in cell rounding and detachment from the tissue culture plastic. We identified the Rho inhibitor p190RhoGAP, a critical regulator of cellular adhesion, as a target of the aberrant kinase.

BCR-ABL nuclear entrapment kills human CML cells: ex vivo study on 35 patients with the combination of imatinib mesylate and leptomycin B.

The BCR-ABL oncoprotein of chronic myelogenous leukemia (CML) localizes to the cell cytoplasm, where it activates proliferative and antiapoptotic signaling pathways. We previously reported that the combination of the ABL kinase inhibitor imatinib mesylate (IM) and the nuclear export inhibitor leptomycin B (LMB) traps BCR-ABL inside the nucleus, triggering the death of the leukemic cells. To evaluate the efficacy of the combination of IM and LMB on human cells we collected CD34-positive cells from 6 healthy donors and myeloid progenitors from 35 patients with CML. The sequential addition of IM and LMB generated the strongest reduction in the proliferative potential of the leukemic cells, with limited toxicity to normal myeloid precursors. Furthermore, nested reverse transcriptase-polymerase chain reaction (RT-PCR) analysis on colonies representative of each experimental condition demonstrated that the combination of IM and LMB was the most effective regimen in reducing the number of BCR-ABL-positive colonies. The efficacy of the 2-drug association was independent of the clinical characteristics of the patients. Our results indicate that strategies aimed at the nuclear entrapment of BCR-ABL efficiently kill human leukemic cells, suggesting that the clinical development of this approach could be of significant therapeutic value for newly diagnosed and IM-resistant CML patients.

The p53-homologue p63 may promote thyroid cancer progression.

Inactivation of p53 and p73 is known to promote thyroid cancer progression. We now describe p63 expression and function in human thyroid cancer. TAp63alpha is expressed in most thyroid cancer specimens and cell lines, but not in normal thyrocytes. However, in thyroid cancer cells TAp63alpha fails to induce the target genes (p21Cip1, Bax, MDM2) and, as a consequence, cell cycle arrest and apoptosis occur. Moreover, TAp63alpha antagonizes the effect of p53 on target genes, cell viability and foci formation, and p63 gene silencing by small interfering (si) RNA results in improved p53 activity. This unusual effect of TAp63alpha depends on the protein C-terminus, since TAp63beta and TAp63gamma isoforms, which have a different arrangement of their C-terminus, are still able to induce the target genes and to exert tumour-restraining effects in thyroid cancer cells. Our data outline the existence of a complex network among p53 family members, where TAp63alpha may promote thyroid tumour progression by inactivating the tumour suppressor activity of p53.

p73 tumor-suppressor activity is impaired in human thyroid cancer.

The p73 protein is a member of the p53 family and, like p53, can induce cell-cycle arrest and apoptosis in response to DNA damage. Because the loss of p53 function is responsible for the progression of well-differentiated thyroid cancer to more aggressive phenotypes, we hypothesized that p73 might also be involved in thyroid carcinogenesis. We find that normal thyrocites do not express p73, whereas most thyroid malignancies are positive for p73 expression. However, the p73 protein of thyroid cancer cells is unresponsive to DNA-damaging agents, failing to elicit a block of the cell cycle or an apoptotic response. Notably, overexpression of transcriptionally active p73 in thyroid cancer lines can arrest the cell cycle but is still unable to induce cell death. The loss of p73 biological activity in neoplastic thyroid cells is partly explained by its interaction with transcriptionally inactive variants of p73 (DeltaNp73) and with mutant p53. Our findings suggest that the functional impairment of p73 could be involved in the development of thyroid malignancies, defining p73 as a potential therapeutic target for thyroid cancer.