The nuclear pore complex (NPC) uses positive residues of amino acids to electrostatically regulate molecular transport through the peripheral route. Xenopus oocyte nucleus to find that excess positive charges of FG-rich nucleoporins slow down passive transport of a polycationic peptide, protamine, without affecting that of a polyanionic pentasaccharide, Arixtra, and small monovalent ions. Protamine transport is slower with a lower concentration of electrolytes in the transport media, where the Debye length becomes comparable to the size of water-filled spaces among the gel-like network of FG repeats. Slow protamine transport is not affected by the binding of a lectin, wheat germ agglutinin, to the peripheral route of the NPC, which is already blocked electrostatically by adjacent nucleoporins that have more cationic residues than anionic residues and even FG dipeptides. The permeability of NPCs to the probe ions is measured by scanning electrochemical microscopy using ion-selective tips based on liquid/liquid microinterfaces and is analysed by effective medium theory to determine the sizes of peripheral and central routes with distinct protamine permeability. Significantly, nanoscale electrostatic gating at the NPC can be relevant not only chemically and biologically, but also biomedically for efficient nuclear import of genetically therapeutic substances.The nuclear pore complex (NPC) is a large protein nanopore that solely mediates molecular transport between the nucleus and cytoplasm of a eukaryotic cell. There is a long-standing consensus that selective transport barriers of the NPC are exclusively based on hydrophobic repeats of phenylalanine and glycine (FG) of nucleoporins. Herein, we reveal experimentally that charged residues of amino acids intermingled between FG repeats can modulate molecular transport through the NPC electrostatically and in a pathway-dependent manner. Specifically, we investigate the NPC of the  Previously, we proposed that the NPC of the Specifically, we find that passive transport of a polycationic peptide, protamine,42, and 10 mM HEPES at pH 7.5  as a negatively charged ionophore to electrostatically bind each guanidinium group of protamine–1 WGA was added to both LSB and MIB solutions (see below). The facilitated transfer of a protamine molecule carrying +20 charges across the interface yields a highly sensitive current response. By contrast, a positively charged ionophore, dimethyldioctadecylammonium (DMDOA+), was added to the NB phase for Arixtra to construct the following cellAg|AgCl|8 μM Arixtra in LSB|5 mM DMDOATFAB and 0.1 M TDATFAB in NB|Agwhere 1.0 g L–1 WGA was also added to LSB (see below). The ammonium group of DMDOA+ electrostatically binds each anionic group of Arixtra to facilitate the electrochemical transfer of –10 charges.e.g., 1 μm in diameter.Specifically, the protamine-selective NB phase was doped with dinonylnaphthalene sulfonate .We employed cyclic voltammetry to ensure high selectivity for protamine in MIB and LSB with physiological and lower ionic strengths, respectively. The potential of the Ag wire in the micropipette was cycled to obtain a current response based on the interfacial transfer of polycationic protamine from LSB to the DNNS-doped NB phase Fig. S2A. The cur Fig. S2B, which d– (0.1 M). A low concentration of 8 μM Arixtra in LSB was detected by using a 3 μm-diameter micropipette , which was ∼200 times in excess with respect to Arixtra.By contrast, an Arixtra-selective current response was obtained with LSB, but not with MIB, which contains a high concentration of interfering Cl Fig. S2C to yieldXenopus oocyte nucleus (∼400 μm in diameter) and spread on the 200 nm-thick microporous region (1.8 mm × 1.8 mm) of a Si3N4 membrane ). The normalized tip current, iT/iT,∞, of 0.80 corresponded to the normalized tip–substrate distance, d/a, of 1.28, i.e. d = 1.9 μm with a = 1.5 μm . By contrast, lower tip currents were obtained at the bottom side of pore 2, which was perhaps blocked by residues of the nucleoplasm. Noticeably, tip currents were slightly higher over the centres of pores 1 and 2 than over those of other pores. This result indicates that the NE was recessed deeper into pores 1 and 2 to increase the tip–NE distance and, subsequently, the tip current. In fact, the ∼100 nm-deep recessed NE in the micropores was observed by AFM.34rotamine  to creati.e., a plot of the tip current, iT, versus the tip–NE distance, d, was measured by vertically bringing a micropipette tip to the centre of a micropore that was uniformly covered with the NE as ensured by SECM imaging, e.g., pores 1, 3, 4, and 5 in k, was assumed to yield the rate of ion transfer across the NE, vNE, as follows:cN and cC are the concentrations of the probe ion at the nucleoplasmic and cytoplasmic sides of the NE, respectively, i.e., in top and bottom solutions in iT/iT,∞versus d/a  with a tip radius of ∼1.5 μm as determined from iT,∞ (eqn (1)) and was related to NPC permeability by effective medium theory,K = 1.8 is the maximum value that corresponds to free diffusion of probe ions through NPCs under a 3 μm-diameter tip (see below).We measured SECM approach curves at micropore-supported NE patches to find that the transport of polycationic protamine through the peripheral region of the NPC is electrostatically blocked in LSB. An SECM approach curve, rsus d/a  to yield2-covered Si wafer), because the NE was perforated by NPCs to transport protamine. Experimental curves (N = 8) fitted well with simulated curves to yield K = 0.2 (red circles) with an uncertainty of 20% caused by non-zero distances (d/a = ∼0.1) at the tip–NE contact.K values of 0.6 (N = 4) and 1.8 (N = 10), respectively. As the KCl concentration changed from 1 mM in LSB to 10 and 100 mM, the ionic strength, I, increased from 7.5 mM to 16.5 and 106.5 mM. The corresponding Debye length, 1/κ, decreased from 3.5 nm to 2.4 and 0.93 nm as given by50ε = 78.49 for diluted aqueous solutions at 25 °C, ε0 is the permittivity of free space, R is the gas constant, and T is the absolute temperature. The ionic strength and Debye length of LSB are limited by 10 mM HEPES and cannot be altered by lowering the KCl concentration. Protamine transport was electrostatically blocked in low ionic strength buffers with 1 and 10 mM KCl, where the Debye length was comparable to the size of water-filled spaces (5.2 nm) among the gel-like network of FG repeats.K value of 1.8.The permeability of the LSB-incubated NE to protamine was lowered by the electrostatic blocking effect from positively charged transport barriers of NPCs. In fact, the tip current became more positive with higher concentrations of 10 and 100 mM KCl  yielded K = 1.8, which corresponds to free diffusion of protamine through the entire pore. In fact, protamine transport in MIB was partially blocked by WGA (blue line) to yield K = 0.2 (N = 9). This low K value is identical to that in LSB, where protamine transfer through the peripheral route was blocked electrostatically. This result confirms that WGA blocked peripheral protamine transport in MIB, where the electrostatic effect was not present.We observed no electrostatic blockage of protamine transport in MIB , which me.g., pores 6 and 7, were selected for approach curve measurements , which was 9 times higher than that of protamine in LSB and was comparable to that of protamine in LSB containing 0.1 M KCl as well as MIB  in comp  is very similar to that of protamine in MIB containing WGA. Importantly, these results ensure that WGA does bind to and block the peripheral route of the NPC in LSB as well as in MIB. Accordingly, the addition of WGA to LSB did not affect the permeability of the NE to protamine  is the membrane porosity. We determined Dw by analysing approach curves at SiO2/Si wafers for polyions  uses and validates γ = 1,6We analysed the permeability of the micropore-supported NE to protamine, Arixtra, and small monovalent ions by effective medium theorypolyions  and 5 anpolyions .32 The f4 cm–1 agrees with the slope determined for these monovalent ions at the intact nucleus of the Xenopus oocyte in MIBr = 24 nm, l = 35 nm, and N = 40 NPCs/μm2 as determined for the NPC of the Xenopus oocyte nucleus.Xenopus oocyte nucleus by using Pt tips, which indicates that tips did not affect NE permeability. Furthermore, the slope of eqn (5), i.e., k/Dw, corresponds to K = 1.8 in eqn (3) with a = 1.5 μm. This K value is limited by free diffusion of a probe ion through the entire pore of the NPC and was obtained for protamine in MIB ), we obtained a linear relationship between the NE permeability and diffusion coefficient for small monovalent ions in LSB and MIB . The sime in MIB  and Arixe in MIB . The corent ions .3 cm–1  with lower protamine permeability in LSB yielded a smaller slope of 2.7 × 10K values of ∼0.2 for protamine (K values of ∼1.8 were obtained for free diffusion of small monovalent ions across the entire pore (Finally, we demonstrate that the sizes of peripheral and central routes are similar in MIB and LSB, thereby confirming that transport barriers of the NPC are not altered in these transport media. Specifically, WGA blocks the peripheral route of the NPC in MIB and LSB to yield similar rotamine  and Arixrotamine , respectire pore . OverallThis work is the first to experimentally prove that molecular transport through the NPC can be electrostatically gated by cationic residues of amino acids intermingled between FG repeats.4i.e., Nup54, Nup58, and Nup62, possess low percentages of amino acids with cationic residues, but a high percentage of FG dipeptides to block passive transport of large substances. In our model,55In this work, we established our model based on the central and peripheral routes of the NPCThere are no conflicts to declare.Supplementary informationClick here for additional data file.


Tumours are complex systems of genetically diverse malignant cells that proliferate in the presence of a heterogeneous microenvironment consisting of host derived microvasculature, stromal, and immune cells. The components of the tumour microenvironment (TME) communicate with each other and with cancer cells, to regulate cellular processes that can inhibit, as well as enhance, tumour growth. Therapeutic strategies have been developed to modulate the TME and cancer-associated immune response. However, modulating compounds are often insoluble (aqueous solubility of less than 1 mg/mL) and have suboptimal pharmacokinetics that prevent therapeutically relevant drug concentrations from reaching the appropriate sites within the tumour. Nanomedicines and, in particular, liposomal formulations of relevant drug candidates, define clinically meaningful drug delivery systems that have the potential to ensure that the right drug candidate is delivered to the right area within tumours at the right time. Following encapsulation in liposomes, drug candidates often display extended plasma half-lives, higher plasma concentrations and may accumulate directly in the tumour tissue. Liposomes can normalise the tumour blood vessel structure and enhance the immunogenicity of tumour cell death; relatively unrecognised impacts associated with using liposomal formulations. This review describes liposomal formulations that affect components of the TME. A focus is placed on formulations which are approved for use in the clinic. The concept of tumour immunogenicity, and how liposomes may enhance radiation and chemotherapy-induced immunogenic cell death (ICD), is discussed. Liposomes are currently an indispensable tool in the treatment of cancer, and their contribution to cancer therapy may gain even further importance by incorporating modulators of the TME and the cancer-associated immune response. Cancer is a leading cause of death worldwide. In 2018, cancer statistics in the United States predicted more than 1.7 million new cancer cases and over 600,000 cancer-related deaths . HoweverTumours have been historically perceived as groups of cells with deregulated growth that proliferate without control and, at later stages, metastasise. However, tumours are not exclusively cells behaving independently and are, instead, complex structures of malignant cells that constantly interact with the surrounding microenvironment  and chanDrug candidates have been developed to target the components of the TME in order to overcome acquired resistances, prevent metastasis of cancer cells, and improve therapeutic efficacy . HoweverThere is a great potential for liposomal formulations to enhance the delivery of compounds with potential anticancer activity—compounds synthesised to modulate the TME and reactivate the tumour-associated immune response. In this review, the main components of the TME and tumour-associated immune response are described, as well as therapeutic approaches to modulate them to achieve improved outcomes in patients with cancer. A search was conducted to identify liposomal formulations that can alter the TME and the immune system. An emphasis was placed on regulatory approved liposomal formulations. The focus, for this reason, is only on liposomal formulations that passively accumulate to regions of the tumour where they can affect the TME. Liposomes actively targeted through tumour-specific ligands or antibodies are described in other reviews ,19, but Tumour cells originate from the accumulation of genetic and epigenetic alterations . These c+-ATPases, the Na+–H+ exchanger NHE1, and the monocarboxylate-H+ efflux cotransporters MCT1 and MCT4 [A primary characteristic of the TME is the presence of hypoxic regions that are less oxygenated and less nutritious relative to normal physiological conditions ; an effeand MCT4 . A direcand MCT4 . Acidityand MCT4 . This aiand MCT4 .From a therapeutic point of view, hypoxia, acidity, and their corresponding modifications in the tumour stroma, can be exploited by nanomedicines, such as liposomes, with an associated drug candidate, to better target the region of cancer growth and widen the therapeutic window. Liposomes that passively accumulate at the tumour site have been designed to activate prodrugs in response to low levels of oxygen and liberate high concentrations of the therapeutic agent in the hypoxic microenvironment. For example, liposomes containing the hypoxia-activated prodrug AQ4N improved cancer treatment outcomes in vivo, preclinically, after photodynamic therapy . LiposomTumours may form new, irregular, and leaky blood vessels that allow the trafficking of tumour-infiltrating lymphocytes (TILs) into the tumour tissue, as if the tissue was undergoing chronic inflammation . HypoxicAngiogenesis occurs by different physiological processes, such as sprouting angiogenesis, intussusceptive microvascular growth, vascular co-option, and vasculogenic mimicry . All theThe leaky blood vessels can be leveraged by certain types of treatments. For instance, high molecular weight drugs and nanoparticles can passively accumulate at the inflamed tumour tissue via the EPR effect . AlternaTumour cells may express neoantigens that can be presented to the immune system, triggering a host immune response . AlthougTumour-associated fibroblasts contribute to remodel the tumour’s extracellular matrix. They are permanently activated and can be differentiated from normal fibroblasts by the production of alpha-SMA and increased expression of fibroblast activation protein (FAP) . Tumour-There is increasing evidence that cells derived exclusively from the adipose tissue (cancer-associated adipocytes) are also involved in remodelling the extracellular matrix . Cancer-As a result of the crosstalk between stromal cells and cancer cells, tumours may exhibit poor perfusion and lack TILs. However, therapeutic strategies have been proposed to induce inflammation in the tumour tissue and trigger lymphocyte infiltration. Intratumour administration of interferon-β, introduction of the TNF superfamily member LIGHT, inhibition of specific oncogenic pathways, and local radiotherapy exhibited improved lymphocyte infiltration in preclinical tumour models . Antican+ T lymphocytes, are correlated with better therapeutic outcomes, and it has been proposed that the presence of these cells can serve as a positive prognostic marker in colorectal cancer [As previously mentioned, genetic mutations that occur in cancer cells result in the generation of neoantigens that can be recognised by the host immune system. In the context of leaky neovasculature and inflammation, immune cells are able to penetrate into the TME and initiate an anticancer immune response. In accordance with this, the presence of TILs, particularly cytotoxic CD8l cancer . Analogol cancer , melanoml cancer , and gasl cancer .+ dendritic cells (first characterised in mice as CD8α+ dendritic cells) acquire antigens from dying tumour cells and target the antigens for cross-presentation via the class I major histocompatibility complex (MHC) processing pathway [+ T-cells and stimulate their infiltration in the tumour. Secretion of type I interferon induced by the STING pathway after detection of extracellular DNA derived from dying tumour cells also seems to have a critical functional role during the antigen cross-presentation to CD8+ T-cells [+ T lymphocytes are then able to recognise cancer cells and eliminate them by releasing perforin and granzymes. Natural killer cells detect and eliminate cancer cells that are deficient in class I MHC, present upregulated stress ligands or are labelled with specific antibodies against tumour neoantigens [The priming of the immune response is a mechanism in which initially DNGR-1 pathway ,62. Dend T-cells . Cytotoxantigens .+ dendritic cells, and infiltration of CD8+ T-cells and NK cells), as suggested above, does not always result in an effective immune response against the tumour. This is mainly due to the aberrant upregulation of a series of immunosuppressive pathways that are necessary for the normal function of the immune system. Several inhibitory pathways have been extensively described, including the programmed death ligand PD-L1/PD-1 pathway [reg) [The presence of all the elements necessary to trigger a host anticancer immune response  ,67,68, aay [reg) . Furtheray [reg) . Tumour-ay [reg) ,72. Myelay [reg) .The blockade of these inhibitory pathways or cell subtypes was conceived as a strategy to block immunosuppression and to reactivate the immune system against the tumour. This has been successfully achieved in the clinic by therapeutic antibodies that are targeted to the ligand PD-L1 , the receptor PD1 , and the receptor CTLA-4 (ipilimumab) . The admIn recent years, immunotherapy has garnered significant attention as it has been achieving great success in the clinic. This is illustrated by treatment approaches, such as blocking immune checkpoints with inhibitors that interfere with the PD-1/PD-L1 interaction to overcome immune suppression, or engineering patient T-cells to recognise and attack cancer cells through CAR-T therapy ,79. GeneICD is a form of cell death where dying tumour cells emit signals known as damage-associated molecular patterns (DAMPs), leading to release of pro-inflammatory cytokines, activation of innate immune cells such as dendritic cells (DCs) and macrophages and, ultimately, stimulation of the adaptive immune response ,86. CurrAs indicated above, a hallmark of ICD is the pre-mortem secretion of ATP. ICD-associated release of ATP involves a complex crosstalk among signalling molecules from multiple distinct cellular processes: autophagy, lysosomal exocytosis, apoptosis, membrane blebbing, and plasma membrane permeabilisation . Upon trRadiotherapy and photodynamic therapy (PDT) have been shown to trigger immunogenic cell death (ICD) within tumours. Radiation therapy is used in the management of approximately 50% of all cancer patients , and is Both tumour cell-intrinsic and extrinsic features promote resistance to radiation therapy. The cell’s intrinsic ability to repair radiation-induced double strand breaks (DSBs) in DNA is key to surviving radiation exposure, and defects in certain DNA repair pathways, such as non-homologous end joining, enhances cellular radiosensitivity . In addiIn addition to radiation, certain chemotherapeutic drugs have been observed to induce ICD. Chemotherapeutic ICD inducers are classified as either Type I or Type II, based on the mechanism by which they induce cell death and ER stress . Type I N,N-diethyl-d-β-γ-dibromomethylene adenosine triphosphate (ARL67156), an extracellular nucleotidase inhibitor, was able to restore ATP secretion and anthracycline-induced ICD in these cells [As described above, there are few existing chemotherapeutics that are bona fide ICD inducers. Furthermore, these agents typically induce ICD as a collateral effect. While the doses being used are sufficient to bring about cancer cell death, the ICD-related effects may be mild or insufficient to stimulate an adaptive immune response, due to dosage, regional exposure to the candidate drug, and/or other factors, such as a poorly immunogenic TME. Many standard care agents are potent drugs that do not trigger ICD, but do emit some DAMPs that are involved in eliciting ICD. For instance, treatment with either cisplatin or gemcitabine is associated with ATP secretion and HMGB1 release, but not cell surface exposure of CRT ,131,132.se cells . This stse cells ,99. All Chemotherapeutics are an invaluable component of many cancer treatment regimes. Unfortunately, treatments that are used clinically are small molecules that may be therapeutically limited by (i) poor aqueous solubility, (ii) rapid depletion from circulation via metabolic and elimination pathways or by poor adsorption, and/or (iii) suboptimal biodistribution properties, which result in significant toxicities and/or the inability of the active agent to reach therapeutically relevant concentrations at the tumour site, at the right time and in the right concentration. By virtue of direct conjugation to change the physical properties of the molecule  or Of the various nanoformulations available, liposomes have emerged as the most clinically successful, in terms of the number of formulations approved , and areGenerally formed from a combination of phospholipids and cholesterol, liposomes are bilayered vesicles in the size range of 50–500 nm. While lipophilic drugs can be incorporated into the lipid membrane, most liposomes contain the majority of their payload encapsulated within the aqueous interior. Drug molecules can be loaded into liposomes passively, via addition during liposome synthesis; this was used as a method to trap cytarabine in Vyxeos™. More commonly, drugs are actively loaded into preformed liposomes using a process that is referred to as remote loading. Drugs cross the lipid bilayer down the drug concentration gradient, then are trapped within the liposome via ionisation as a result of a high or (more commonly) low pH interior environment , or via A further advantage to the use of liposomes as anticancer drug delivery vehicles is that they provide opportunities for surface functionalisation. The circulation half-life of liposomes can be further prolonged through the incorporation of polyethylene glycol (PEG) chains into the lipid bilayer . The PEGLiposomal encapsulation has eliminated the need of toxic excipients in the formulation of poorly water-soluble compounds, such as paclitaxel , and altFirst termed by Maeda in the 1980s , the enh(1) Nanoparticle pharmacokinetics. Small molecule chemotherapeutic drugs and peptides generally have short in vivo half-lives as a result of poor physical and metabolic stabilities coupled with rapid renal and hepatic eliminations. Association of a drug molecule with a nanoparticle, however, confers to it the properties of the nanoparticle system which are designed to be retained in the plasma compartment for extended time periods following intravenous administration. The relatively large nanoparticle (10 nm to 200 nm) and its associated corona (particle with bound proteins) can often-times shield the associated drug from the in vivo environment. As such, while the drug is still associated with the nanoparticle, it is often still in an active form, and is available for therapeutic activity once dissociated from the nanoparticle.(2) Tumour vasculature. The rapid growth of solid tumours requires the delivery of extra nutrients that the body’s normal vasculature cannot provide. As such, tumours orchestrate the formation of their own system of blood vessels in a process known as angiogenesis (see above). The rapid propagation of tumour-associated blood vessels results in chaotic and poorly formed “leaky” vessels. The long circulation time of nanoparticles means that they have multiple exposures to the tumour vasculature, and they can extravasate from the blood through leaky vessels into the TME over time.(3) Impaired lymphatic drainage. In normal physiology, the lymphatic network of vessels returns fluid and large molecules that have extravasated into tissue, back into the systemic circulation. The high interstitial pressure of the TME, in conjunction with rapidly growing cells, puts increased pressure on tumour lymphatic vessels, impairing their functionality. The presence of lymphatic vasculature within solid tumours is variable, and those found in the TME are significantly impaired . ExtravaChemotherapy generally follows a treatment schedule designed to deliver a high dose of drug, and a recovery period prior to subsequent doses to allow healthy cells/tissues to recover. However, it has also been suggested that within this recovery period, cancer and stromal cells, and specifically vascular endothelial cells, also have the opportunity to recover . “MetronLiposomal doxorubicin was the first of many liposomal anthracycline formulations approved for the clinical treatment of cancer. It was interesting that Doxil™ was first approved for AIDS-related Kaposi’s sarcoma, which is an endothelium-related disease, yet the antiangiogenic activity of encapsulated doxorubicin was only recently reported. Initial studies in murine orthotopic models of glioblastoma indicated, via MRI imaging, the incidence of haemorrhaging within the tumours of mice administered multiple doses of PEGylated liposomal doxorubicin, whereas no haemorrhaging was observed in mice administered saline or free doxorubicin . While tAnother interesting example concerns tumours initiated following injection of a P-glycoprotein (Pgp)-upregulated colorectal cancer cell line. These cells showed resistance to doxorubicin in vitro; however, the same cells grown in vivo demonstrated comparable growth delay patterns to the wild-type cell line following the administration of liposomal doxorubicin. The discrepancy between in vitro and in vivo results was shown to be at least partly caused by increased vascular cell death in mice bearing the doxorubicin-resistant cell line when compared to the wild-type tumours. It was suggested that the liposomal doxorubicin facilitated the death of vascular endothelial cells. This, in turn, reduced the growth rate of the tumour in vivo, demonstrating the importance of vascular cell death in tumour growth .+) cells in subcutaneous colorectal tumours of mice treated weekly for 6 weeks, when compared to saline-treated mice [Tumour normalisation effects have been reported following administration of liposomal irinotecan. A liposomal formulation of irinotecan (Irinophore C™) decreased both the density of tumour cells and tumour vasculature , liposomal vincristine, or liposomal doxorubicin (Doxil™) all reduced the growth of a subcutaneous murine model of glioblastoma, and increased the percentage of perfused tissue per tumour when compared to the saline control . FurtherIt was shown that irinotecan, doxorubicin, and vincristine are all potent against vascular cells in culture, when under proliferative conditions versus non-proliferative conditions. As such, if these drugs are released from liposomes in the TME, it is possible that they can act on proliferating  blood vessels . Based oAdditional studies sought to assess the efficacy of 5-FU administered following treatment and vasculature normalisation with Irinophore C™. Irinophore C™ treatment lead to the initial decrease in vascular function; however, after 3 rounds of chemotherapeutic treatment, the percentage of vascular cells in tumours were significantly increased when compared with saline-treated control mice . Irinoph®-solubilised paclitaxel against a human melanoma cell line, it had an improved in vivo activity against a humanised model of melanoma in SCID mice. Further, the liposomal formulation, but not the Cremophor® formulation, reduced tumour vessel density in this in vivo model [While not yet approved in American or European countries, a liposomal paclitaxel formulation has been approved for the treatment of ovarian cancer, breast cancer, and non-small cell lung cancer in China. In a series of studies, the Dellian group demonstrated the antivascular effects of paclitaxel when delivered in a cationic liposomal formulation. Initial studies demonstrated that, although this liposomal formulation of paclitaxel showed comparable in vitro efficacy with Cremophorvo model . The grovo model . Contribvo model . Furthervo model .®-solubilised paclitaxel, the changes in tumour growth rates achieved with the liposomal formulation could be attributed to multiple tumour and TME factors. The group briefly examined co-administration of unencapsulated cisplatin with the paclitaxel liposomes, and they demonstrated a further improvement in in vivo efficacy [Taken together, these studies demonstrate the capacity of paclitaxel to directly interact not only with tumour cells, but also alter the tumour vasculature. While liposomal paclitaxel had greater efficacy in treating subcutaneous melanoma tumours when compared to results in animals treated with Cremophorefficacy , however+ T-cells, NK cells). It is important to recognise that liposomes, like all nanomedicines, are known to be eliminated, in part, by cells of the mononuclear phagocyte system (MPS); mainly monocytes and macrophages [The slow release of drug(s) from liposomes mimicking metronomic dosing will also modulate the behaviour and content of immune cells present in the TME, although this has yet to be optimised. Ideally, a nanomedicine should have an enhanced inhibitory effect on immunosuppressor cells  and engender higher activation of immune cells that promote antitumour effects  to boost the antitumour response of different cancer immunotherapies . First otibodies . Liposom+ tumour-associated macrophages, implying that the increased accumulation of drug in the tumour was not related to the uptake of liposomal drug by macrophages, but to alternative mechanisms known to enhance tumour vascular permeability [+ T-cells and Fas-mediated death, as demonstrated in vitro [+ and CD8+ T-cells, and reduced myeloid-derived suppressor cells not only in the treated tumour, but also in the contralateral tumour site. All these observations link use of liposomal doxorubicin to immune mediated effects on the tumour.The effects of liposomal doxorubicin were also studied on tumour-associated macrophages . Liposomeability . Anothereability . Howeverin vitro . In the in vitro . The comMifamurtide is muramyl tripeptide phosphatidylethanolamine (MTP-PE), a lipophilic synthetic derivative of a peptide naturally occurring in the bacterial wall of the bacteria Bacillus Calmette–Guerin. Mifamurtide, associated with liposomes, administered to osteosarcoma patients in combination with chemotherapy, had improved survival in comparison to patients that only received chemotherapy . In cont+ cells, tentatively suggesting that radiation-induced enhancement of antigen-presentation was promoting a synergistic response with the liposome-based tumour vaccine [Few studies have directly assessed the ability of liposome-delivered agents to synergise with radiation and promote ICD. One strategy reported by Chamoto et al., used liposomes to co-encapsulate a tumour-specific peptide (OVA) and the TLR9 agonist CpG, a commonly used vaccine adjuvant . Intrade vaccine . Importa vaccine . In addi vaccine ,183, hig2) tumour cells to ionising radiation in vitro, and the authors provided some evidence that these liposomes relieved tumour hypoxia in vivo, whereas catalase-free Pt (IV)-liposomes had no effect on tumour hypoxia [Other strategies to enhance radiation-induced ICD involve targeting features of the TME that limit radiation therapy’s cytotoxicity, such as hypoxia . In some hypoxia . Ultimat hypoxia . The stu hypoxia ,189, an The success of nanomedicinces, in particular, liposomal drugs and drug combinations, has been discussed above. The advantage of these formulations likely involves their ability to alter the pharmacokinetic profile and biodistribution of the associated drug(s). As a consequence of this treatment, efficacy can be enhanced and the formulations can exhibit reduced toxicity. At the time that this review is written (2018), the use of nanoparticles to induce ICD has not been thoroughly explored. Nanoparticles are known to interact with the immune system and can be engineered to attain the desired immunomodulatory effects ,191. It Liposomal formulations that are currently used in the clinic were reported to reduce the formation of tumour neovasculature in the TME, as illustrated by studies with liposomal doxorubicin, liposomal irinotecan, and liposomal paclitaxel. However, the research investigating the effects of liposomes on the tumour vasculature is still scarce. It should be noted that some drugs have been described as inhibitors of tumour neovasculature (e.g., daunorubicin  and vincTherapeutic antibodies acting against angiogenesis were also coupled to liposomes. Researchers prepared bevacizumab–liposome conjugates to prolong the residency of bevacizumab in the vitreous after intravitreal administration . In anotClinical liposomal formulations were also studied for their positive modulation on the immune cells present in the TME. Liposomal doxorubicin and liposomal mifamurtide enhanced the host immune response in different fashions. Do liposomal cytarabine, daunorubicin, vincristine, and irinotecan have an effect on the immune components of the TME? Liposomes have also been targeted to macrophages by several mechanisms, including peptides, antibodies, and lectins . In precLiposomes are a tool to deliver molecules to regions of cancer growth, either through slow release of drug from liposomes in the blood, or release of liposomes that have accumulated in tumours via the EPR effect. Liposomal formulations of drugs have translated into increased efficacy in killing tumour cells, but also affecting different tumour-promoting cell subtypes in the TME, such as vascular endothelial cells, regulatory T-cells, and other cells from the myeloid linage. Three liposomal formulations used to treat cancer in humans were reported to inhibit angiogenesis in the TME: liposomal doxorubicin, liposomal irinotecan, and liposomal paclitaxel. Furthermore, two approved liposomal formulations were reported to enhance tumour-associated immune responses: liposomal doxorubicin and liposomal mifamurtide. Even though research involving liposomes and ICD is still limited, we believe that liposomes are best suited to enhance radiation and chemotherapy-induced ICD. In the latter case, research efforts should be put, first, at identifying drug combinations effective at triggering immunogenic death of tumour cells. At a later step, such agents should be co-encapsulated into liposomes to combine the synergistic immune effects of both agents with the classical benefits associated when using liposomal formulations. There are several liposomal formulations that have shown therapeutic benefits by modulating the TME and tumour-associated immune response. Further research will allow the identification of liposomal formulations and combinations, thereof, with improved efficacy against cancer.


Primary central nervous system (CNS) solitary fibrous tumour/hemangiopericytoma (SFT/HPC) is a rare neoplasm and its classification criteria have been redefined by the latest WHO Classification of CNS Tumours. Outcome can vary significantly among patients, thus reliable prognostic markers are warranted.Primary CNS SFT/HPC diagnosed at the Pathology Unit of our Institution between 2006 and 2016 were retrospectively collected. Tumour grade along with immunohistochemistry for Ki67, STAT6, PHH3, CD34 and Bcl-2 were assessed. TERT promoter status was evaluated by Sanger sequencing.Fifteen SFT/HPC were analysed: 9/15 (60%) female, median age at diagnosis 60 (range: 10–67). Six (40%) cases showed a SFT phenotype and mean H&E-mitotic count was 4.8/10 HPF. Tumour grade was I in 6, II in 4 and III in 5 cases. Mean PHH3-mitotic count was higher than H&E count (8.4 versus 4.8/10 HPF), but it would have determined a change in tumour grade in a sole case. Nuclear staining for STAT6 was present in 14/15 (93.3%). CD34 and Bcl-2 expression rates were lower in higher grade tumours. TERT promoter was mutated in two cases. Median follow up time was 2.4 years (6 months-7.4 years) and 5/15 (33%) patients developed local disease recurrence. Partial resection (p = 0.0185), higher WHO grade (p = 0.038), lower CD34 (p = 0.038) and Bcl-2 (p = 0.010) expressions were significantly associated with a poorer disease-free interval.WHO grade is the main prognostic tool in CNS SFT/HPC, but it could be integrated by other markers, like CD34 and Bcl-2, in the clinical practice. The relevance of TERT promoter mutations in this subset of CNS tumours needs further evaluation. NAB2) and the signal transducer and activator of transcription 6 (STAT6) genes , 3. This alteration is indeed present in most SFT/HPC cases regardless of tumour site or grade. NAB2-STAT6 fusion protein promotes tumour growth by localizing to the nucleus and activating the EGR gene. Nuclear localization of the fusion protein can be detected by anti-STAT6 antibodies[Classification of primary CNS solitary fibrous tumour/hemangiopericytoma (SFT/HPC) has been recently redefined by the latest World Health Organization (WHO) Classification of Tumours of the Central Nervous System (CNS) by mergintibodies and STATntibodies, 4CNS SFT/HPC are rare , usually dural-based intracranial tumours, thought to develop from CD34-positive fibroblasts present in the dura mater or in the intraparenchymal perivascular connective tissue, 6 and mPrognosis is often good, especially in cases with the solitary fibrous tumour morphology and when gross total resection is achieved; conversely, the hemangiopericytoma morphology is associated with a higher rate of local recurrences (>75% in patients with long term follow up) and distant metastases occurrence.–12 BasedTERT gene codes for the catalytic subunit of the telomerase complex and recurrent point mutations of its promoter have been shown to create a novel binding site for E-twenty-six (ETS) family transcription factors, leading to increased telomerase activity in affected cells. TERT promoter mutations are common to different malignancies, with variable prognostic significance.[Overall, compared to extra-CNS SFT/HPC, prognostic markers are less defined and available data are limited by the rarity of this tumour; thus, new and reliable prognostic factors are warranted to help personalize patients’ therapeutic management. Therefore, we wanted to retrospectively assess novel possible prognostic factors in a series of SFT/HPC diagnosed at our Institution, evaluating both histopathological and molecular features. In particular, we evaluated the prognostic relevance of two IHC markers: CD34 and B-cell lymphoma 2 (Bcl-2), which, although non-specific, can be useful diagnostic tools and were especially important in the pre-STAT6 era. CD34 losificance.–22 Sinceificance. we wanteFifteen consecutive cases of primary CNS solitary fibrous tumours or hemangiopericytomas diagnosed at the Pathology Unit of City of Health and Science University Hospital of Turin between 2006 and 2016 were retrospectively collected. All patients underwent surgical resection at the Neurosurgery Unit of the same Institution. Follow up data were retrieved from patients’ charts. The study was conducted in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans and within guidelines and regulations by the Research Ethics Committee of the University of Turin. Written informed consent was not obtained because patients were no longer undergoing regular follow up visits directly at our institution or were deceased; considered the retrospective nature of the present study and that it had no impact at all on patients’ care, verbal informed consent was obtained from participants or their relatives and recorded in a specific log. This study, including the verbal consent procedure, was approved by the Research Ethics Committee of the University of Turin.All diagnoses were confirmed by a senior neuropathologist (PC) and reassessed according to the 2016 WHO classification. Thus, tumours were graded based on their phenotype (solitary fibrous tumour versus hemangiopericytoma) and mitotic count on H&E slides. Mitotic count was repeated after PHH3 IHC . IHC for STAT6 , Ki67 , CD34  and Bcl-2  were also performed. Immunohistochemistry for Ki67, CD34 and Bcl-2 was performed on all tissue blocks of each case; Ki67 labelling index was evaluated by counting at least 1000 tumour cells while CD34 and Bcl-2 were assessed by visual estimation. Nuclear staining for STAT6 was considered consistent with the SFT/HPC diagnosis. BenchMark ULTRA platform  was used for all IHC.DNA extraction from formalin-fixed and paraffin-embedded (FPPE) tumour samples was performed as previously described and concMutational status of the telomerase reverse transcriptase (TERT) promoter region from position -27 to -286 from ATG start site, including the polymorphic site represented by rs2853669, were determined by PCR and Sanger sequencing using the following primer pair: promoter forward 5′-CAGCGCTGCCTGAAACTC-3′ and reverse 5′-GTCCTGCCCCTTCACCTT-3′, as described by Horn et al. PCR prod2 test. Kruskall-Wallis has been used to compare not normally distributed variables. The variables tested were: age at diagnosis, gender, tumour phenotype, grading based on H&E and on PHH3 mitotic count (<5/10 HPF versus ≥5/10 HPF), tumour size based on MRI report before surgery (<3 cm versus ≥3), Ki67 labelling index (≤5% versus >5%), CD34 expression (<80% versus ≥80%), Bcl-2 expression (<30% versus ≥30%), TERT promoter status and tumour recurrence. Differences were considered significant when p < 0.05 for reported two-sided p-values. Survival curves between different groups were plotted using the Kaplan-Meier method and the statistical comparisons were performed with Log-rank test.Statistical analyses were performed using Stata/MP 15.0 Statistical Software . Categorical variables were compared using Pearson’s χFifteen SFT/HPC cases were retrospectively collected: 9/15 (60%) were female, median age at diagnosis was 60 (range: 10–67). Median follow up time was 2.4 years (6 months-7.4 years). Gender, age and tumour site were not significantly correlated with tumour morphology/phenotype . Tumour Six cases showed a solitary fibrous tumour phenotype  . Mean nuAlthough the mean number of mitoses/10 HPF observed following PHH3 IHC staining was higher than in H&E  , in one Nuclear staining for STAT6 was present in 14/15 (93.3%) . The onlTERT promoter was successfully analysed in twelve cases out of 15 (80%) . TERT promoter resulted wildtype in 10/12 (83%) cases, while 2/12 (17%) were mutated: the recurrent 1,295,228 C>T (C228T) mutation and a 1,295,254 C>T (C254T) mutation positioned respectively at 124 and 150 base pairs upstream of the ATG translational start site of TERT were identified; these mutations were found in a grade I and a grade II SFT/HPC, respectively. The first patient had no disease recurrence (follow up: 73 months), while the second patient had no recurrence too, but died of unrelated causes 5 months after diagnosis. Six out of 12 (50%) cases harboured the rs2853669 single nucleotide polymorphism (SNP). Neither of these findings was associated with tumour phenotype (p = 1.000 and p = 0.248) or DFI (log-rank test p = 0.387 and p = 0.739).The identification of prognostic markers is a crucial issue for appropriate patients’ management, even more when it applies to rare entities or after changes in the classification criteria. Since SFT/HPC of the CNS well resume both these issues, in the present study we analysed and identified putative prognosticators which could be used in the daily practice. Prognostic factors are especially needed in this setting considered that some of the parameters used for risk-stratification of extra-CNS SFT/HPC, like tumour size, are not applicable in the CNS.Beginning with clinical variables, GTR was significantly associated with a better DFI, a finding consistent with literature data., 12, 27 Focusing on histopathological features, the two different tumour phenotypes (SFT and HPC) were not specifically associated with clinical variables : as a matter of fact, a distinction based on their clinico-radiological characteristics was not deemed possible even when they were considered different entities. The HPC In our series, PHH3-based mitotic count was higher than the H&E-based (as expected), but it would have changed the WHO grade (from grade II to grade III) in one case, only; moreover, grading based on this tool was not associated with a different DFI, therefore suggesting its lack of utility in this specific subset of tumours. These results need to be validated in a larger cohort, but for the time being the use of PHH3-based mitotic count in daily practice is not supported.Among other IHC markers, STAT6 nuclear staining was detected in all cases except for one 14/15, 93.3%), a finding consistent with literature data., 93.3%, , 31 and TERT promoter mutations have been identified in CNS SFT/HPC, but specific data in this setting are limited.[versus 25.6%), a difference even greater when considering the HPC phenotype alone (16.7% versus 50%).[TERT promoter mutational status and outcome, but this could probably be explained by the limited sample size. Moreover, one of the two identified mutations (C254T) was still unreported in this tumour entity. We also investigated a common TERT promoter SNP (rs2853669): its frequency in our series (50%) is in line with its prevalence in the general population and we did not observe an association with outcome. limited., 33 In osus 50%). We did nTERT promoter mutations, could provide valuable information, but their significance in this setting needs further evaluation in larger series.In conclusion, WHO grade is the main prognostic tool for CNS SFT/HPC, but our data suggest that it could be integrated in the daily practice by other markers, like CD34 and Bcl-2. Also molecular traits, like


Herein, we first went through basic principles and applications of thiol-reactive based probes in protein imaging and recognition. We then summarize a family of metal-NTA based fluorescence probes in the visualization of His6-tagged protein and identification of metalloproteins at proteome-wide scale. The pros and cons of the probes, as well as ways to optimize them, are discussed.Fluorescent probes such as thiol-reactive and Ni Because of their relatively small sizes, these probes have less perturbation on the investigated proteins -complex, i.e., FLAsH-EDT2 (EDT = ethanedithiol), to site-specifically recognize proteins containing four cysteines at the i, i + 1, i + 4, and i + 5 positions of an α-helix in 1998 -based probes was designed and further modified for 2 and ReAsH-EDT2 are turn-on fluorescent probes as the 1,3,2-dithiarsolane (EDT) results in weak fluorescence before thiol-reaction with cysteines. Such a benefit was inherited in the design of other As-based probes, NPE, and CTNPE. Due to the structural features of vicinal-dithiol-containing proteins (VDPs), only one As(III) was kept for protein recognition, whilst two biocompatible diglycol amine groups were integrated into these probes to improve their water solubility and biological compatibility -based fluorescent probes, FLAsH-EDTe et al. . The tetnC- rather than tetracysteine. Acting as the reductive end of this oxidation-reduction network, VDPs can be labeled by NPE both in vitro and in vivo. Staining of NPE finally lit up the VDPs in live cells without invasiveness Ni- and red (NTA-AB)Ni- probes could overcome the cellular intrinsic autofluorescence with better contrast than the blue probe, NTA-ACNi-. Moreover, these probes can be used to quantify His6-tagged proteins with a wider linear range  compared with Western Blot , where M stands for metals  for protein identification. The identified proteins were then subjected to Gene Ontology (GO) enrichment analysis and mapped into protein networks. This approach implements the exploration of endogenous metal-associated proteins in various biological systems, including those proteins that bind metal ions weakly or transiently , while asparagic acid 63 (D63), tyrosine 95 (Y95), and lysine 206 (K206) participate in the formation of H-bonding to stabilize the structure. Interestingly, the probe folds a sandwich-like structure and Fe3+ coordinates to only two carboxylates and one nitrogen of the probe and one oxygen from Y188, leaving enough vacant site for interacting with proteins  was firstly mined and 44 Ni2+-associated proteins were identified , a keystone species to trigger periodontitis  fluorophores became a fast-growing and exciting field because of low absorption through biological matrixes -based probes entered cells successfully whilst an array of Ni-NTA-based probes lack cellular membrane permeability. Despite diverse structure optimizations on these Ni-NTA-based probes, no clear clue of cellular membrane permeability was presented. Consequently, the different permeability between these two classes of probes resulted in their different applications in biology besides labeling relevant peptide-fused proteins. As(III)-based probes light up the native As(III)-binding proteins -based probes and metal-NTA-based probes, were introduced and summarized from their binding mechanisms, molecule structures, to biological applications. These probes initially targeted fused proteins with either tetracysteine or His2+ was selected as a showcase to inherit the success of Ni-NTA-based probes. In total, three probes were synthesized, i.e., NTA-ACNi-, NTA-AFNi-, and NTA-ABNi-, with fluorescent emission ranging from blue to red. Importantly, they can track intracellular His-tagged proteins in live cells  can label the relevant metal-associated proteins through metal ion-oriented recognition. As a result, the networks of Ni2+, Bi3+, Cu2+, and Fe2+/3+-associated proteins (and their proteomes) have been lit up in situ and in vivo (Lai et al., Our probes were built upon metal-NTA coordination chemistry, and NiNJ drafted this manuscript. HL revised this whilst. HS supervised this.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The regulation of ubiquitination status in the cell is controlled by ubiquitin ligases acting in tandem with deubiquitinating enzymes. Ubiquitination controls many key processes in the cell from division to death making its tight regulation key to optimal cell function. Activity based protein profiling has emerged as a powerful technique to study these important enzymes. With around 100 deubiquitinating enzymes and 600 ubiquitin ligases in the human genome targeting a subclass of these enzymes or even a single enzyme is a compelling strategy to unpick this complex system. In this review we will discuss different approaches adopted, including activity-based probes centered around ubiquitin-protein, ubiquitin-peptide and mutated ubiquitin scaffolds. We examine challenges faced and opportunities presented to increase specificity in activity-based protein profiling of the ubiquitin conjugation/deconjugation machinery. Also, the presence of a native isopeptide bond in the scissile position may affect probe stability in complex systems.A further example of this probe type was developed by the Chatrerjee laboratory in 2016 using a selenocystine ligation  to sequentially functionalize thiols at a single carbon center for profiling E1 enzyme activity (Stanley et al., 73-thioester was reacted with azidoaminoethane to afford Ub-azide. Alkyne functionalized acrylate and acrylamide were used to prepare two TDAE functionalized ubiquitin monomers. A single cysteine mutant of His-UBE2L3 was reacted with the monomers to form the E2-Ub conjugate probes (Elaborated TDAE probes aimed to specifically target the E3 ligase Parkin by incorporating ubiquitin into the probe (Pao et al., e probes . The trie probes  later sh75-NH2NH2 using native chemical ligation followed by Dha formation. The probes were tested against catalytic domains of NEDD4 and UBE3C, labeling both active sites. Probing HeLa cells saw enrichment of several E3 enzymes, with strong enrichment of NEDD4. This work demonstrates an alternative route to E2-Ub probes however the linker is three atoms longer than the native and the electrophilic trap is presented two and five atoms away from Ub and UBE2D2, respectively.Shi and co-workers (Xu et al., Several approaches have been taken to confer selectivity to activity-based probes for ubiquitin conjugation/deconjugation machinery . MutatioCertain conjugate probes are limited due to their hydrolysable linkers and despite several well-designed solutions, many of the probes also do not perfectly mimic the linker length or trap position of the wild-type substrate. There is therefore scope for optimisation of the probe design and implementation of new chemistry for the synthesis of novel probes. Furthermore, these probes are currently limited by their lack of cell permeability. Recent work demonstrated that incorporation of cleavable cell-penetrating peptides can help deliver monoubiquitin probes into a cell (Gui et al., JM and NT drafted and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Personalized hydration strategies play a key role in optimizing the performance and safety of athletes during sporting activities. Clinicians should be aware of the many physiological, behavioral, logistical and psychological issues that determine both the athlete’s fluid needs during sport and his/her opportunity to address them; these are often specific to the environment, the event and the individual athlete. In this paper we address the major considerations for assessing hydration status in athletes and practical solutions to overcome obstacles of a given sport. Based on these solutions, practitioners can better advise athletes to develop practices that optimize hydration for their sports. Maintaining euhydration, the state of preserving body water within its optimal homeostatic range, is essential to sustain life. Water contributes 50–70% of total body mass and is compartmentalized within both intracellular 65%) and extracellular 35%) spaces . Euhydra5% spaces5% and exWhile the sensation of thirst, a centrally mediated response to body water deficits, is useful in dictating the need for fluid intake during daily life, thirst is relatively insensitive in acutely tracking hydration status during exercise ,19. MainCurrent consensus recommends that good hydration practices include: (1) beginning exercise in a state of euhydration, (2) preventing excessive hypohydration during exercise, and (3) replacing remaining losses following exercise prior to the next exercise bout ,20,21. THydration assessment can be utilized to indicate one’s current hydration state, but if taken serially, it can also be used to track changes in hydration and indicate fluid needs . While various methods of hydration assessment exist, there is no single method that can serve as a criterion measure to assess hydration status in all settings . Plasma osmolality, changes in plasma volume, and the volume, osmolality and specific gravity of urine are the most commonly published metrics to assess changes in hydration status in clinical settings . The useIn field applications, the careful assessment of changes in body mass over a bout of physical activity provides a reasonably accurate assessment of body water deficits incurred during the session, since sweat loss and fluid intake during the session underpin the major changes in body mass and body water content. This is true for most sporting activities conducted over a duration of <2–3 h; however, during very prolonged and strenuous exercise , other factors that cause mass changes, metabolic water production and the liberation of stored water become numerically important and undermine the utility of this assessment ,27,28. AA useful paradigm for tracking daily changes in hydration status in sporting situations is to consider a combination of assessments to track daily changes. The monitoring of daily changes in body mass, coupled with urine color and thirst sensation status provides adequate sensitivity for most athletic situations . CheuvroPractical Solutions:(1)Carefully monitor acute changes in body mass over an exercise bout to determine sweat rate, adequacy of fluid replacement and fluid needs for recovery for that session. Consider how well this can be used to evaluate general hydration strategies in similar situations.(2)Use changes in body mass, urine color and thirst upon awakening to track daily changes in hydration status.Body fluid loss during sport or exercise largely results from sweating. Net fluid balance is modulated to a certain extent by drinking. Rate of sweating is primarily a function of metabolic heat production , but canExercise intensity is the main factor that determines metabolic heat production, meaning that the rate of fluid losses from sweat for a given exercise session can be partially explained by the intensity of the exercise . Total fFor sports like cycling and running, the influence of exercise intensity and duration on fluid needs is very easy to determine given the consistent nature of exercise. 2max), gastric emptying decreases predictably, most likely based on decreased splanchnic perfusion [It should also be noted that exercise intensity influences gastric emptying rate . Individerfusion .Practical Solutions:(1)Increased fluid intake is necessary with prolonged or intense exercise due to increased sweat production.(2)2max) understand that gastric emptying may limit fluid absorption. Athletes can train their gut to improve gastrointestinal comfort or adopt strategies to increase fluid intake before and after exercise.During vigorous exercise  exposure tends to increase water and electrolyte losses, decrease plasma volume and total body water content . In bothPractical Solutions:(1)Measure local environmental conditions to determine the risk of high sweat rates resulting in large fluid losses.(2)Increase fluid-replacement during exercise in hot and humid environments to account for increased sweat losses.(3)Account for clothing or equipment requirements when evaluating fluid needs.(4)Modify fluid intake when exercising in cold or altitude according to an estimation of fluid losses noting that thirst may be less reliable as a guide to dehydration under these conditions.Fluid availability refers to the factors that dictate an athlete’s ability to replace fluid losses during activity. In many instances, the characteristics of the sport have a strong influence on the ability to drink during competition and in many scenarios prevent an athlete from “drinking to thirst” . MeanwhiIn endurance sports which provide competitors with feed zones/water stations , the number of water stations on a course and the frequency with which a competitor reaches them can influence drinking behavior. The International Association of Athletics Federations recommends that stations are placed approximately every 5 km, however, many races include more frequent stations which may influence athletes’ drinking strategies and behaviors . Slower The official rules and competition characteristics of “stop-start” sports such as team and racket sports create other influences, and often unique scenarios, around fluid availability. In some examples , governing rules limit the availability of fluids for athletes during competition. Soccer, for example, includes two 45-min halves (with a continuous running clock) in which fluid availability is extremely limited to players. At the other end of the spectrum are sports such as baseball, basketball and tennis with frequent rest breaks within playing time  during which fluids can be consumed. An athlete’s drinking strategy for a competition represents a unique instance for their particular sport based on their ability to rehydrate within the rules . We suppIn Practical Solutions:(1)During training, ensure that there is ample access to fluids that are palatable to athletes.(2)Investigate or understand the opportunities for fluid intake during that are specific to a sport or event, and any other practical issues that determine fluid intake.(3)Consider the risks of hyperhydration as well as hypohydration for any sporting event or individual athlete, and prepare appropriate practice and education strategies.(4)Develop personalized fluid intake plans that incorporate fluid availability characteristics of the sport or event. Where there is a likelihood of hypohydration, be proactive and creative in making use of existing opportunities for fluid intake within sport rules and characteristics and be prepared to request for changes when there is a likelihood of a serious mismatch between fluid losses and the opportunity to address these.(5)Practice intended competition drinking plans ahead of time to determine their suitability and allow time for readjustment.A number of intrinsic factors modulate the individual variances that are observed in fluid losses. One of the greatest considerations for an individual’s sweat rate is his or her body size. Larger individuals typically have higher sweat losses, with football linemen exhibiting some of the highest recorded sweat rates . TherefoHeat acclimatization contributes to variations in an individual’s sweating rate responses. Individuals who are heat acclimatized exhibit greater sweat rates which can pose a greater risk of hypohydration . AlthougWomen may be at greater risk for exercise-induced hyponatremia. This risk has been attributed to their lower body weight and size, excess water ingestion, and longer racing times relative to men . The grePractical Solutions:(1)Consider body size, acclimatization status and thirst drive when developing hydration plans for individual athletes.The culture and normal behaviors surrounding specific sports can greatly affect the hydration practices of its athletes. The three most prominent examples of the cultural effects of sports on hydration practices are weight division sports, acrobatic sports and appearance-based sports. In weight division sports , the practice of deliberately dehydrating to manipulate body mass to meet lighter competition weight classifications is common . In manyPractical Solutions:(1)Promote healthy weight management strategies in weight division, acrobatic and appearance-based sports that minimize manipulation of body water.Based on the factors in the above sections, along with published literature on typical fluid balance observations in various sports ,63, we aIn this paper we present a paradigm that can be used by clinicians and practitioners to develop hydration strategies for sports based on fluid availability, environment and exercise intensity. These tools are provided to inform hydration education and practices in a dynamic and individualized manner so that athletes can adapt to different circumstances and optimize performance.


Drosophila and thus they may also impact honey bee queen fertility and health. However, the specific molecules in semen and seminal fluid that initiate post-mating changes in queens are still unidentified. Herein, we summarize the mating biology of honey bees, the changes queens undergo during and after copulation, and the role of drone semen and seminal fluid in post-mating changes in queens. We then review the effects of seminal fluid proteins in insect reproduction and potential roles for honey bee drone seminal fluid proteins in queen reproduction and health. We finish by proposing future avenues of research. Further elucidating the role of drone fertility in queen reproductive health may contribute towards reducing colony losses and advancing honey bee stock development.Honey bees are major pollinators of agricultural and non-agricultural landscapes. In recent years, honey bee colonies have exhibited high annual losses and commercial beekeepers frequently report poor queen quality and queen failure as the primary causes. Honey bee colonies are highly vulnerable to compromised queen fertility, as each hive is headed by one reproductive queen. Queens mate with multiple drones  during a single mating period early in life in which they obtain enough spermatozoa to fertilize their eggs for the rest of their reproductive life span. The process of mating initiates numerous behavioral, physiological, and molecular changes that shape the fertility of the queen and her influence on the colony. For example, receipt of drone semen can modulate queen ovary activation, pheromone production, and subsequent worker retinue behavior. In addition, seminal fluid is a major component of semen that is primarily derived from drone accessory glands. It also contains a complex mixture of proteins such as proteases, antioxidants, and antimicrobial proteins. Seminal fluid proteins are essential for inducing post-mating changes in other insects such as  Apis mellifera) colonies in the United States alone are valued at $14.6 billion annually [Varroa destructor as the two most common reasons for colony losses in the past several years [Insect pollination of commercial crops is valued worldwide at $175 billion annually and pollination services provided by commercially managed honey bee  and undertake one or more nuptial flights before they cease performing mating flights . Once thThe ovaries of queens are essential for egg production and are approximately eight times bigger in mated, egg-laying queens compared to virgin queens ,73. MatiE)-9-keto-2-decenoic acid (9ODA), -(−)- and -(+)-9-hydroxy-2-decenoic acid (9HDA), methyl p-hydroxybenzoate (HOB), and 4-hydroxy-3-methoxyphenylethanol (HVA) [The best-studied glands are the queen’s mandibular glands which are large sacs attached to the mandibles and generate queen mandibular pheromone (QMP), which is primarily composed of five chemicals: (ol (HVA) ,54,74,77ol (HVA) ,81,82,83ol (HVA) ,81,82,83ol (HVA) ,81,82,83ol (HVA) ,84. Inteol (HVA) . Thus, pol (HVA) .The Dufour’s gland also elicits retinue responses in workers and may signal ovary activation when produced in workers ,86. The After copulation, queens exhibit transcriptional and protein-level changes in the spermatheca, ovaries, and brain ,89,90,912), a substance commonly used to sedate queens during instrumental insemination and also happens to induce queen ovary activation, causes effects similar to mating by inducing changes in gene expression in the brains of queens, such as reduced expression of cognition and vision-related genes [Bombus terrestris) and distantly related Drosophila melanogaster regulate expression of vision-related genes involved in phototransduction after mating [In addition, brain gene expression and sexual receptivity are correlated ,68. Inteed genes ,93. Reprr mating ,95. Thus2, queens exhibit decreased brain gene expression of dopamine receptors and activation of ovaries [2 exposure [Mating also alters the morphology of the brain  and resu ovaries . In contexposure . This suexposure . In summary, there is good empirical evidence that copulation alters numerous biological aspects of queen reproduction. These now well-characterized post-mating changes are important and quantifiable proxies that can be used for testing the effects of different copulation factors on queen post-mating changes.2 [2 exposure, physical manipulation of the vaginal tract, insemination volume, and insemination fluid composition  also differentially affects brain and ovary gene expression and has a greater impact on ovary gene expression than insemination volume, as queens inseminated with either 1 μL or 8 μL of semen had more similar ovary expression profiles than queens inseminated with saline of either volume . However2 exposure . Queensn review ). They an review ). Furthen review ).Drosophila and other insects has determined that female post-reproductive changes can be largely attributed to the receipt of seminal fluid proteins rather than other mating components [The aforementioned studies indicate that the processes involved in queen post-mating changes are complex and are differentially affected by numerous factors such as insemination volume and insemination fluid composition. Honey bee seminal fluid contains proteins that likely serve as key drivers of seminal fluid-dependent post-mating changes in queens. Substantial work in mponents ,108.Drosophila females, where mated females exhibit shorter lifespans, lower metabolism, decreased receptivity to mating, increased oviposition rates, increased expression of immune-related genes, and overall differential gene expression compared to virgin flies [Drosophila and increasingly studied and supported in other insects, including mosquitos, crickets, ants, moths, and beetles [Drosophila has identified specific proteins that reduce female sexual receptivity , maintain spermatozoa viability , promote uterine contractions , and ovulation . IntereTo date, mass spectrometry has been used to identify the proteins in honey bee drone semen , accessoHoney bee SFPs encompass several different biological pathways, including reactive oxidative species defense  and metabolism , suggesting their roles in protecting spermatozoa against oxidative damage and supporting spermatozoa metabolism ,148. IndNosema apis [N. apis spore viability via two respective mechanisms [Nosema spore walls, whereas the non-protein fraction of seminal fluid directly decreases spore viability without cell wall rupture or spore germination [N. apis spores, which indicates there are multiple seminal fluid proteins with antifungal activity [Several immune-related proteins are also found in honey bee seminal fluid, including chitinases, Osiris 7, and heat shock proteins, which have been associated with immune defense against pathogens ,160,161.ema apis ,148. Botchanisms . Seminalmination . When seactivity . ComponeDrosophila [Nosema spp., have been detected in queens , so even highly infected drones are physically able to travel to DCAs and potentially mate and infect queens [V. destructor mites, a major vector of Deformed Wing virus, exhibit decreased flight ability and spermatozoa counts [N. apis and Nosema ceranae have also been detected in honey bee semen [N. ceranae exhibit altered flight patterns but are able to maintain spermatozoa viability [It remains to be studied whether drone seminal fluid proteins also exhibit antibacterial or antiviral activity, but the honey bee seminal fluid proteome includes several heat shock proteins, which are important for antiviral immunity in osophila  and likeosophila ,159,161.osophila . Althougosophila ,165, sevreviewed ). Virusereviewed  and queeed areas ,166,167.a counts . Most noee semen , even thee semen . Drones iability . N. apis exhibit differential expression of 111 seminal fluid proteins, a large proportion of which are involved in immunity and detoxification [Nosema infection alters pheromone production in queens [N. apis also causes differences in other reproductive changes in queens. In addition, since viruses and V. destructor are prevalent throughout colonies and infect/infest drones, it will be important to determine how they alter seminal fluid composition and subsequent queen reproductive changes and health. Drones infected by fication . The antfication . As Nosen queens , it woulThere is now solid evidence that seminal fluid proteins are key drivers of post-reproductive changes in many species, and it is likely that honey bee drone seminal fluid proteins also influence post-mating changes in queens ,148. FurThe queen is an important member of the honey bee colony and can be a major determinant of colony health and productivity. Drones, too, are very important players as they can have a strong impact on queen post-mating changes and subsequent colony health. Specifically, drone seminal fluid modulates queen sexual receptivity, pheromone production and seminal fluid proteins are likely the key drivers of these changes ,106. In In addition, drone seminal fluid proteomes vary based on genetic lineage . Based oN. apis [Teleogryllus commodus and Acheta domesticus, prostaglandins present in their seminal fluid are responsible for reducing sexual receptivity and inducing oviposition in recipient females [Furthermore, it is important to reiterate that honey bee seminal fluid is composed of both proteins and unidentified non-protein components, which are likely peptides, lipids, and sugars ,115. In N. apis , the non females ,176. Pro females , but it V. destructor-resistant honey bees that exhibit higher expression of proteins associated with V. destructor resistance [Lastly, identifying the specific roles of SFPs in queen reproduction could have an important impact on improving bee breeding practices in order to develop more resilient genetic honey bee stock. For example, being able to manipulate the production of specific SFPs in drones could lead to improved queen reproductive fitness particularly in breeder queens. Such selective breeding practices were utilized to develop sistance ,178. Ult


HeteroGenesis simulates more complex and realistic heterogeneous tumour genomes than existing methods, can model different evolutionary dynamics, and enables the creation of multi-region and longitudinal data.Tumour evolution results in progressive cancer phenotypes such as metastatic spread and treatment resistance. To better treat cancers, we must characterize tumour evolution and the genetic events that confer progressive phenotypes. This is facilitated by high coverage genome or exome sequencing. However, the best approach by which, or indeed whether, these data can be used to accurately model and interpret underlying evolutionary dynamics is yet to be confirmed. Establishing this requires sequencing data from appropriately heterogeneous tumours in which the exact trajectory and combination of events occurring throughout its evolution are known. We therefore developed HeteroGenesis: a tool to generate realistically evolved tumour genomes, which can be sequenced using weighted-Wessim (w-Wessim), an https://github.com/GeorgetteTanner, implemented in Python and supported on linux and MS Windows.HeteroGenesis and w-Wessim are freely available under the GNU General Public Licence from Bioinformatics online. The evolution of a tumour affects its clinical course: malignant phenotypes undergo selection resulting in increased growth, invasion, metastases or therapy resistance. Elucidating the mode and course of tumour evolution therefore impacts upon our understanding of disease progression and how to more effectively treat patients . Numerouin silico whole exome sequencing (WES) tool, Wessim  to be incorporated into the genomes for each clone in a tumour, along with a matched germline. It takes as input: (i) a reference FASTA genome sequence, (ii) an optional file containing known germline SNV and InDel locations and minor allele frequencies formatted from dbSNP and (iii) a JSON file containing a set of parameters. It outputs a JSON file with lists of variants for each clone in the simulated tumour and a matched germline, as well as files containing the order that mutations occurred. heterogenesis_varincorp is run separately for each clone, and the germline sample. It takes the lists of variants generated by heterogenesis_vargen and incorporates them into the reference genome sequence, as well as calculating copy numbers and variant frequencies along the genome. freqcalc is then run to combine variant profiles for individual clones from heterogenesis_varincorp outputs to describe one or more bulk samples.HeteroGenesis consists of three consecutive python programmes  include simulated germline sequencing data to mimic the contamination of normal cells in a tumour sample, (ii) create multiple bulk datasets from the same tumour with different proportions of clones to mimic multi-region sampling, an approach that enables more reliable delineation of subclone architectures  as the probes in the BLAT alignment. By filtering these reads for those with a high alignment mapping score and adjusting the stringency of the BLAT alignment, we are able to generate reads with w-Wessim that match the distribution of reads seen in the real data, with 79.7% of bases aligning on or near target and a mode coverage of 28x . A demonbty1063_Supplementary_DataClick here for additional data file.


The occurrence of these slow earthquakes is discontinuous along the trench and attributed to the effect of high pore pressures at the plate boundary. Here, we show that spatial variations in intraslab stress may control fluid migration from the subducted Philippine Sea slab to the plate boundary. The triaxial normal faulting stress, detected by stress tensor inversion using focal mechanisms in the slab, controls anisotropically permeability that trends NNW–SSE subhorizontally from the subducted Philippine Sea slab to the plate boundary. The inhomogeneous permeability controlled by spatial stress heterogeneities in the subducted Philippine Sea slab controls the intraslab fluid pathway. This hypothesis is consistent with the spatial heterogeneity of slow earthquakes and  Water in the subducting plate is released into the overlying mantle wedge by the dehydration of hydrous minerals in subducting oceanic crust2. At some island arcs, a contrast in permeability across the Moho results in the accumulation of water and the build-up of high pore fluid pressure in the corner of the mantle wedge overlying the subducting plate3. A discontinuous band of slow earthquakes along the Nankai subduction zone is observed in the Kii channel, which is located between the Kii Peninsula and the Shikoku district in Japan 7 to separate stresses derived from earthquake focal mechanism data from the spatially variable state of stress.We used focal mechanism data of seismic events in the Philippine Sea slab beneath the Shikoku district and the Kii Peninsula to infer the present-day intraslab stresses Fig. . Recent Figure 1 and σ3 orientations (strike/dip) of 153°/55° and 245°/2°, respectively, and a stress ratio Φ (Φ = (σ2 - σ3)/(σ1 - σ3)) of 0.92 15, where μ is the friction coefficient and λ is the pore fluid pressure ratio. We classified the 36 earthquake events into three groups as F1, F2, and F3 to estimate μ′ for each fault group compatible only with the stress. Assuming a constant friction coefficient μ = 0.616, the effective friction coefficients of groups F1, F2, and F3 were calculated as 0.02, 0.01, and 0.08, respectively  within a stress regime are represented by Mohr circlesely Fig. . The coe18 and from seismic tomography19. The intraslab earthquakes activated by the normal faulting stress (Stress C) are dominant in the region of convex-shaped slab  is confined to a limited part of the slab. Third, the pore fluid pressure ratio is high in the slab. We propose that the significant stress contrasts in the Philippine Sea slab cause regional variations in fluid migration within the slab. The permeability of rocks may be related to the σram Fig. . The sta3He/4He ratio distribution in the Kii Peninsula and western and central parts of Shikoku, which are regions of anomalous 3He/4He ratios. Recent studies27 have reported that the 3He/4He ratios in these regions show anomalously high values despite the fore-arc location, and the high ratios may be attributable to mantle helium derived from a magma source28. The inferred anisotropy of permeability in the region underlying the Kii Peninsula and western and central Shikoku is associated with high 3He/4He ratios. We are now able to explain, in terms of the heterogeneous distribution of stresses in the slab, why the region with a high 3He/4He ratios occur not only along the Kii Peninsula but also across the entire peninsula and western and central parts of Shikoku. Hence, the characteristic fluid migration mechanisms associated with Stress C are more favourable for generating fractures, which in turn facilitate the migration of mantle-derived helium.The anisotropy of intraslab permeability causes regional variations in fluid migration in the Philippine Sea slab. This inference is consistent with the λ = 0.85–0.97) inferred by the present study is equivalent to a pore fluid pressure ratio λ = 0.9529 under the conditions of stress and temperature at depths where intraslab earthquakes occur. In the Nankai subduction zone, the fluids are liberated from the subducting slab at depth of 30–60 km30. This depth almost overlaps the depth of the earthquakes in the slab crust and mantle we used. If the spatial heterogeneity in fluid migration from the slab produces regional variations in the fluid volume, the accumulation of fluid and the build-up of high pore fluid pressure in the corner of the mantle wedge vary spatially along the slab. A relatively low value (λ = 0.85) of pore fluid pressure ratio in the slab with Stress C may support a large amount of dehydration to mantle wedge compared to the slab with Stresses A and B. We infer that slow earthquakes linked along dip in the Nankai subduction zone4 occur in the corner overlying the region of subducting plate where Stress C dominates. Stress C is not dominant in the Kii channel where slow earthquake activity is extremely low4 (Fig. 3He/4He ratio distribution (Fig. The anisotropy of permeability caused by Stress C promotes fluid migration trending NNW–SSE subhorizontally. The high pore fluid pressure ratio (7 to the data shown in Fig. 15 with a resampling technique for separating stresses from heterogeneous focal mechanism data7. Significant stresses are represented as clusters of reduced stress tensors and are identified by k-means clustering31.We applied the MIM


To prove the potential applicability of the fluid in in vitro bioaccessibility test, we compared bioaccessibility of particulates-bound trace elements using this fluid with those of stimulated epithelial lung fluid. Bioaccessibility were relatively low values (<30%) in locus bean substituted lung fluid and stimulated epithelial lung fluid. Specifically, As and Cd had significantly higher bioaccessibility values in locus bean substituted lung fluid than stimulated epithelial lung fluid. The data demonstrate that fluid formulated and used in this study can provide a suitable means of evaluate bioaccessibility of trace elements-bound to airborne particulates.This study was piloted to evaluate bioaccessibility of particulate-bound trace elements using synthetic epithelia lung fluid; in which dipalmitoylphophatidylcholine was substituted with locus bean gum (LBSFL). The resulting data reveal that no significant change in physicochemical characteristics of the stimulated lung fluid compare with similar synthetic fluids; pH value of 7.3, density (0.998gcm•The fluid was used for assessing bioaccessibility of particulate matters-bound trace elements•The formulated fluid can be applied to study in toxicity assessment•The data can be used for inter-laboratory comparison of bioaccessibility of particulate -bound trace element and could stimulate environmental concerns on the impacts of airborne particulates. Specifications TableIn risk assessment, one of the challenges for environmental toxicologist has been development of fluid with properties similar to human tracheobronchial fluids , so as t2SO4), sodium chloride (NaCl), CaCl2, NaHPO4, NaHCO3, KCl, MgCl2, were purchased from Merck, London. Potassium Chloride (KCl), were obtained from BDH. Ascorbic acid, uric acid, glutathione, albumin, cysteine, glycine and polyethylene oxide resin were products of Sigma-Aldrich. All experimental solutions were prepared using double deionized water .All chemicals used in this work were of analytical grade and were used without further purification. Glycine and cysteine were purchased from Sigma Aldrich, UK. Anhydrous sodium sulphates (NaInstruments used in this study included the Inductively Coupled Plasma – Mass Spectrometry (ICPMS), Sciex Elan DRC II, which was operated in standard mode equipped with a Meinhard Concentric Quartz Nebuliser, cyclonic spray chamber, and platinum skimmer and sampler cones.•1000 ml of the fluid was prepared by separate addition of the inorganic phase to organic phase.•2, 150 mg NaHPO4, 2700 mg NaHCO3, 298 mg KCl, 200 mg MgCl2, 72 mg Na2SO4 into a 500 mL HDPE and made up to the volume with ultra-pure water, and then thoroughly mixed.The inorganic phase was prepared by accurately weighed: 6020 mg NaCl, 256 mg CaCl•Similarly, the organic phase was prepared by accurately weighed: 18 mg ascorbic acid, 16 mg uric acid and 30 mg glutathione into a 500 mL HDPE and made up to the volume with ultra-pure water, and then thoroughly mixed.•The two (inorganic and organic) phases were poured into 2 L conical flask containing 260 mg albumin, 122 mg cysteine, 100 mg locus bean gum, 376 mg glycine and 500 mg mucin.•The contents were thoroughly mixed to dissolve all the components.•The physical characteristics of the fluid; pH meter , density (Pycnometer) , conductIn this study, the recipe and procedures for preparation of locus bean substituted fluid was based on those of previously formulated stimulated epithelial lung fluids described in . Table 1in vitro bioaccessibility extraction of particulates-bound trace elements. For this purpose, dataset of total suspended particulate matters collected from different locations at Ado-Ekiti (The formulated LBSLF was used in do-Ekiti  were use0C (1-h shaking followed by 1 h still). The obtained extracts were cooled to room temperature, then centrifuged for 20 min at 3500 rpm and separated for element determination. The percentage bio accessible fraction was calculated as follows [For bioaccessibility study, one quarter of the filter membranes used in samples collection was placed into three different 15 ml polypropylene centrifuge tubes, containing 10 ml each of formulated LBSLF, stimulated epithelia lung fluid (SELF) and artificial lysosome fluid (ALF). The tubes were made airtight and immersed for 2 h in a shaking water bath at 37 follows :(1)PerceThe elemental content of the particulates were determined by Inductively Coupled plasma mass spectrometer (Aligent 5973 inert) (ICPMS). The operating conditions are: plasma flow (18.0 L/min), auxiliary flow (1.80 L/min), Nebulizer flow (1.07 L/min), sheath depth (0.30 L/min), sampling depth (5.00 min), stabilization delay (10 s), power (1.40 kW), pump rate (5 rpm).For quality control, a standard reference material, NIST SRA 1649(Urban particulate matter) was analyzed along with blank filter. The detection limits for the methods were calculated by multiplying the standard deviation of the blank results by 3; in cases where more than one blank was used for an extraction. The concentrations of elements were calculated after subtraction of the blank filter concentration from total concentration in the particulate matters. The recovery of elements was determined by the addition of a spiking solution (prepared using deionized water and standard solution) containing 0.5 mg/L of As, Cd, Cu, Mn, Ni, Zn, Pb were conducted on LBSLF with and without sample. The percentage recovery levels were within 100 ± 15% in all the steps.−3) was almost close to 0.998gcm−3 previous formulation. The conductivity of 13.9 mS m-1 is higher than the reference solution (water) but comparable with 14.5 ± 0.1 mS m-1 previously reported [-1, is slightly lower than 55 mN m-1 reported in earlier study and 72 mN m-1 of reference liquid (water). The use of locust bean gum serves to improve the surface tension of formulated fluid in the present work. The observed high conductivity of the fluid compared to water has been attributed to tonicity effects of the dissolved inorganic salts [The importance of fluids composition on bioaccessibility of different elements has been stretched in several reports . The usereported . Viscosiic salts . Althoug2.5 was considered in this study, as this is the size that can penetrate deeply to the respiratory airways. The bioaccessibility of elements as percentage of element extractable with the formulated fluid in various sites was determined in this study. Only PM