Recommendations for blood sampling in emergency departments from the European Society for Emergency Medicine (EUSEM), European Society for Emergency Nursing (EuSEN), and European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group for the Preanalytical Phase. Executive summary.

AIM: Blood Sampling Guidelines have been developed to target European emergency medicine-related professionals involved in the blood sampling process (e.g. physicians, nurses, phlebotomists working in the ED), as well as laboratory physicians and other related professionals. The guidelines population focus on adult patients. The development of these blood sampling guidelines for the ED setting is based on the collaboration of three European scientific societies that have a role to play in the preanalytical phase process: EuSEN, EFLM, and EUSEM. The elaboration of the questions was done using the PICO procedure, literature search and appraisal was based on the GRADE methodology. The final recommendations were reviewed by an international multidisciplinary external review group. RESULTS: The document includes the elaborated recommendations for the selected sixteen questions. Three in pre-sampling, eight regarding sampling, three post-sampling, and two focus on quality assurance. In general, the quality of the evidence is very low, and the strength of the recommendation in all the questions has been rated as weak. The working group in four questions elaborate the recommendations, based mainly on group experience, rating as good practice. CONCLUSIONS: The multidisciplinary working group was considered one of the major contributors to this guideline. The lack of quality information highlights the need for research in this area of the patient care process. The peculiarities of the emergency medical areas need specific considerations to minimise the possibility of errors in the preanalytical phase.

Improved cytometric analysis of untouched lung leukocytes by enzymatic liquefaction of sputum samples.

BACKGROUND: Phenotyping sputum-resident leukocytes and evaluating their functional status are essential analyses for exploring the cellular basis of pathological processes in the lungs, and flow cytometry is widely recognized as the gold-standard technique to address them. However, sputum-resident leukocytes are found in respiratory samples which need to be liquefied prior to cytometric analysis. Traditional liquefying procedures involve the use of a reducing agent such as dithiothreitol (DTT) in temperature-controlled conditions, which does not homogenize respiratory samples efficiently and impairs cell viability and functionality. METHODS: Here we propose an enzymatic method that rapidly liquefies samples by means of generating O2 bubbles with endogenous catalase. Sputum specimens from patients with suspected pulmonary infection were treated with DTT, the enzymatic method or PBS. We used turbidimetry to compare the liquefaction degree and cell counts were determined using a hemocytometer. Finally, we conducted a comparative flow cytometry study for evaluating frequencies of sputum-resident neutrophils, eosinophils and lymphocytes and their activation status after liquefaction. RESULTS: Enzymatically treated samples were better liquefied than those treated with DTT or PBS, which resulted in a more accurate cytometric analysis. Frequencies of all cell subsets analyzed within liquefied samples were comparable between liquefaction methods. However, the gentle cell handling rendered by the enzymatic method improves cell viability and retains in vivo functional characteristics of sputum-resident leukocytes (with regard to HLA-DR, CD63 and CD11b expression). CONCLUSION: In conclusion, the proposed enzymatic liquefaction method improves the cytometric analysis of respiratory samples and leaves the cells widely untouched for properly addressing functional analysis of lung leukocytes.

Respuesta patológica completa tras quimioterapia neoadyuvante en cáncer de colon localmente avanzado / Complete pathologic response after neoadjuvant chemotherapy in locally advanced colon cancer

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N,N-bis-heteroaryl methylamines: Potent anti-mitotic and highly cytotoxic agents.

The synthesis and evaluation of a series of N,N-bis-heterocyclic-methylamines 1 as isoazaerianin analogues are described. It was demonstrated that the replacement of the 3,4,5-trimethoxyphenyl A-ring present in CA-4, isoCA-4 and isoazaerianin by a quinoline or a quinazoline ring is possible and often beneficiary for a high level of cytotoxicity. We have also showed that a carbazole or an indole nucleus are very effective as B-rings in this series, leading to anti-cancer drugs 1 having a sub-nanomolar level of cytotoxicity (1a: IC50 = 70 pM against HCT116 cells). 1a also display a high level of cytotoxicity against four other human cancer cells and inhibited tubulin assembly at a micromolar level. Moreover, at a concentration of 5 nM, 1a arrested the cellular cycle in G2/M phase of the cellular cycle and induced apoptosis of HCT116 cells. It was also showed that after few hours 1a at a concentration of 10 nM totally disrupted endothelial network formation on Matrigel.

Initial stages of development of the submandibular gland (human embryos at 5.5-8 weeks of development).

The aim of this study was to determine the main stages of submandibular salivary gland development during the embryonic period in humans. In addition, we studied submandibular salivary gland development in rats on embryonic days 14-16 and expression in the submandibular salivary gland region with the monoclonal antibody HNK-1. Serial sections from 25 human embryos with a greatest length ranging from 10 to 31 mm (Carnegie stages 16-23; weeks 5.5-8 of development) and Wistar rats of embryonic days (E) 14-16 were analysed with light microscopy. Five stages of submandibular salivary gland development were identified. The prospective stage (1), between weeks 5.5 and early week 6, is characterized by a thickening of the epithelium of the medial paralingual groove in the floor of the mouth corresponding to the primordium of the submandibular salivary gland parenchyma. At this stage, the primordium of the parasympathetic ganglion lies below the lingual nerve. The primordium of the submandibular salivary gland parenchyma is observed in rats on E14 in the medial paralingual groove with mesenchymal cells, underlying the lingual nerve. These cells are HNK-1-positive, corresponding to the primordium of the parasympathetic ganglion. The bud stage (2), at the end of week 6 in humans and on E15 in rats, is characterized by the proliferation and invagination of the epithelial condensation, surrounded by an important condensation of the mesenchyme. The pseudoglandular stage (3) at week 6.5 is characterized by the beginning of the formation of lobes in the condensed mesenchyme. The canalicular stage (4), between week 7 and 7.5, is characterized by the appearance of a lumen in the proximal part of the submandibular duct. The innervation stage (5) occurs during week 8, with the innervation of the submandibular and interlobular ducts. Nervous branches arriving from the parasympathetic ganglion innervate the glandular parenchyma. Numerous blood vessels are observed nearby. Our results suggest that submandibular salivary gland development requires interactions among epithelium, mesenchyme, parasympathetic ganglion and blood vessels.

N-myristoyltransferases inhibitory activity of ellagitannins from Terminalia bentzoë (L.) L. f. subsp. bentzoë.

N-myristoylation (Myr) is an eukaryotic N-terminal co- or post-translational protein modification in which the enzyme N-myristoyltransferase (NMT) transfers a fatty acid (C14:0) to the N-terminal glycine residues of several cellular key proteins. Depending on the cellular context, NMT may serve as a molecular target in anticancer or anti-infectious therapy, and drugs that inhibit this enzyme may be useful in the treatment of cancer or infectious diseases. As part of an on-going project to identify natural Homo sapiens N-myristoyltransferase 1 inhibitors (HsNMT1), two ellagitannins, punicalagin (1) and isoterchebulin (2), along with eschweilenol C (3) and ellagic acid (4) were isolated from the bark of Terminalia bentzoë (L.) L. f. subsp. bentzoë. Their structures were determined by means of spectroscopic analyses and comparison with literature data. Punicalagin (1) and isoterchebulin (2) showed significant inhibitory activity towards HsNMT1, and also against Plasmodium falciparum NMT (PfNMT) both in vitro and in cellulo, opening alternative paths for new NMT inhibitors development. This is the first report identifying natural products from a botanical source as inhibitors of HsNMT and PfNMT.

Inhibition of p53-Murine Double Minute 2 (MDM2) Interactions with 3,3'-Spirocyclopentene Oxindole Derivatives.

3,3'-Spirocyclopentene oxindoles structurally related to Wang's spiropyrrolidine oxindoles have been highlighted as a new class of antiproliferative agents against cancer cell lines with wild-type p53 status (IC50 up to 0.96 µM on SJSA-1 and 2.9 µM in HCT116 p53-wt). Inhibition of the MDM2-p53 interactions has been demonstrated through in vitro HTRF assays (IC50 up to 3.1 nM), while Western blot analysis showed activation of p53 selectively in HCT116 cancer cell lines with wild-type p53.

mTOR Inhibition via Displacement of Phosphatidic Acid Induces Enhanced Cytotoxicity Specifically in Cancer Cells.

The mTOR is a central regulator of cell growth and is highly activated in cancer cells to allow rapid tumor growth. The use of mTOR inhibitors as anticancer therapy has been approved for some types of tumors, albeit with modest results. We recently reported the synthesis of ICSN3250, a halitulin analogue with enhanced cytotoxicity. We report here that ICSN3250 is a specific mTOR inhibitor that operates through a mechanism distinct from those described for previous mTOR inhibitors. ICSN3250 competed with and displaced phosphatidic acid from the FRB domain in mTOR, thus preventing mTOR activation and leading to cytotoxicity. Docking and molecular dynamics simulations evidenced not only the high conformational plasticity of the FRB domain, but also the specific interactions of both ICSN3250 and phosphatidic acid with the FRB domain in mTOR. Furthermore, ICSN3250 toxicity was shown to act specifically in cancer cells, as noncancer cells showed up to 100-fold less sensitivity to ICSN3250, in contrast to other mTOR inhibitors that did not show selectivity. Thus, our results define ICSN3250 as a new class of mTOR inhibitors that specifically targets cancer cells.Significance: ICSN3250 defines a new class of mTORC1 inhibitors that displaces phosphatidic acid at the FRB domain of mTOR, inducing cell death specifically in cancer cells but not in noncancer cells. Cancer Res; 78(18); 5384-97. ©2018 AACR.

A New Series of Succinimido-ferrociphenols and Related Heterocyclic Species Induce Strong Antiproliferative Effects, Especially against Ovarian Cancer Cells Resistant to Cisplatin.

Ferrociphenols are known to display anticancer properties by original mechanisms dependent on redox properties and generation of active metabolites such as quinone methides. Recent studies have highlighted the positive impact of oxidative stress on chemosensitivity and prognosis of ovarian cancer patients. Ovarian adenocarcinomas are shown to be an excellent model for defining the impact of selected ferrociphenols as new therapeutic drugs for such cancers. This work describes the syntheses and preliminary mechanistic research of unprecedented multitargeting heterocyclic ferrociphenols bearing either a succinimidyl or phthalimidyl group that show exceptional antiproliferative behavior against epithelial ovarian cancer cells resistant to cisplatin. Owing to the failure of the present pharmaceutical options, such as carboplatin a metallodrug based on Pt coordination chemistry, these species may help to overcome the problem of lethal resistance. Currently, ferrociphenolic entities generally operate via apoptotic and senescence pathways. We present here our first results in this new cyclic-imide series.

Original Vinca Derivatives: From P-Glycoprotein Substrates to P-Glycoprotein Inhibitors.

The first example of vinca derivatives 16-18 able to modulate P-glycoprotein (Pgp) efflux activity is reported. They were elaborated in two steps from vinorelbine 3 (VLN) by a modification of the velbenamine moiety. These compounds were able to decrease efficiently Pgp mediated influx and efflux of rhodamine-123 (Rho) and to restore the cytotoxicity of vinorelbine 3 (VLN) and doxorubicin (Dox) on K562R (dox-resistant) cell lines.

Synthesis and biological evaluation of C-13' substituted 7'-homo-anhydrovinblastine derivatives.

Recent publications highlighted that vinca derivatives either functionalized on C-12' or enlarged on cycle C' could be more cytotoxic than vinblastine or vinorelbine, both used in anti-cancer therapy. By combining these two results, nine new 7'-homo-anhydrovinblastine derivatives functionalized on C-13' were elaborated. The synthesis of key intermediates, their one-step transformation into final products in mild conditions and their biological activities are presented.

One-pot synthesis of vinca alkaloids-phomopsin hybrids.

Hybrids of vinca alkaloids and phomopsin A have been elaborated with the aim of interfering with the "vinca site" and the "peptide site" of the vinca domain in tubulin. They were synthesized by an efficient one-pot procedure that directly links the octahydrophomopsin lateral chain to the velbenamine moiety of 7'-homo-anhydrovinblastine. In their modeled complexes with tubulin, these hybrids were found to superimpose nicely on the tubulin-bound structures of vinblastine and phomopsin A. This good matching can account for the fact that two of them are very potent inhibitors of microtubules assembly and are cytotoxic against four cancer cell lines.

A concise formation of N-substituted 3,4-diarylpyrroles--synthesis and cytotoxic activity.

A short synthesis of N-substituted 3,4-diarylpyrroles by condensation of a phenacyl halide with a primary amine and a phenylacetaldehyde is reported. The key step is an intramolecular cyclization of an in situ generated enamine onto a ketone. Using differently substituted aromatic reactants and N-(3-aminopropyl)azatricyclodecane as the amine component, the preparation of analogs of the cytotoxic marine alkaloid halitulin could be achieved. The cytotoxicity of some of the compounds obtained by this method was studied, and one of them proved to be a very potent derivative, acting at a nanomolar concentration, in a caspase-independent cell death mechanism.

Synthesis and biological evaluation of a new series of highly functionalized 7'-homo-anhydrovinblastine derivatives.

Sixteen new 7'-homo-anhydrovinblastine derivatives were prepared in one or two steps from vinorelbine by means of an original and regiospecific rearrangement and subsequent diastereoselective reduction. This strategy has allowed fast access to a family of vinca alkaloid derivatives with an enlarged and functionalized ring C'. Their synthesis and biological evaluation are reported. One compound (compound 35) is 1.7 times more active than vinorelbine as a tubulin assembly inhibitor. Moreover, some of these compounds are highly cytotoxic, and two of them are more potent than vinorelbine on HCT116 and K562 cell lines. Molecular modeling studies, carried out with two of the new vinca derivatives, provide useful hints about how a given functionalization introduced at positions 7' and 8' of the C' ring results in improved binding interactions between one of the new derivatives and the interdimer interface when compared to the parent compound vinblastine.

UPLC-ESI-MS analysis of thymosins ß4 and ß10 in cell lysates: a simple, rapid and sensitive quantification method.

Fast and reproducible quantification of thymosins ß4 and ß10 in different cell cultures was achieved by ultra high performance liquid chromatography coupled to mass spectrometry. We demonstrated that cancer cell lines all exhibit a higher amount of Tß10 compared to control cells, whereas the level of Tß4 is drastically depending on cell lines.

Overexpression of the natural tetrapeptide acetyl-N-ser-asp-lys-pro derived from thymosin beta4 in neoplastic diseases.

The natural tetrapeptide acetyl-ser-asp-lys-pro (AcSDKP) is formed in vivo by enzymatic cleavage of the N terminus of thymosin beta4 by prolyl oligopeptidase (POP). Recently, AcSDKP was shown to promote angiogenesis. Because of the critical role of neovascularization in cancer development, the levels of AcSDKP and POP activity in a number of different malignant tissues were investigated. Our studies revealed that AcSDKP levels were markedly elevated in neoplastic diseases including hematologic malignancies and solid neoplasms. Consistent with this finding, the enhanced activity of POP was also detected in all analyzed specimens of cancer tissues. Both these novel findings are in concert with the previously reported overexpression of thymosin beta4 in a large variety of malignant tumors and with its potential role in cancerogenesis. The physiological relevance of these findings awaits further studies; however, our first results strongly suggest a key role for AcSDKP in the pathogenesis of cancer.

N-Terminal 2,3-diaminopropionic acid (Dap) peptides as efficient methylglyoxal scavengers to inhibit advanced glycation endproduct (AGE) formation.

2,3-Diaminopropionic acid (Dap) and N-terminal Dap peptides have been found to inhibit in vitro protein-modifications by methylglyoxal (MG), one of the highly reactive alpha-dicarbonyl compounds. MG scavenging potency of the newly synthesized N-terminal Dap peptides is demonstrated by RP-HPLC, SDS-PAGE and non-denaturing PAGE analysis, assays for enzymatic activity and cell viability study and was compared with that of known AGE inhibitors, such as aminoguanidine, pyridoxamine, metformin and carnosine. Two addition products of MG and L-Dap-L-Leu are separated by HPLC and their chemical structures are characterized by (1)H and (13)C NMR spectroscopy to indicate that both of them are pyrazines derived from 2 molecules of MG and 1 molecule of L-Dap-L-Leu. Mutagenic activities of L-Dap-L-Leu and L-Dap-L-Val and their metabolites according to the Ames assay are found to be negative.

In vitro and in vivo properties of ellagic acid in malaria treatment.

Malaria is one of the most significant causes of infectious disease in the world. The search for new antimalarial chemotherapies has become increasingly urgent due to the parasites' resistance to current drugs. Ellagic acid is a polyphenol found in various plant products. In this study, antimalarial properties of ellagic acid were explored. The results obtained have shown high activity in vitro against all Plasmodium falciparum strains whatever their levels of chloroquine and mefloquine resistance (50% inhibitory concentrations ranging from 105 to 330 nM). Ellagic acid was also active in vivo against Plamodium vinckei petteri in suppressive, curative, and prophylactic murine tests, without any toxicity (50% effective dose by the intraperitoneal route inferior to 1 mg/kg/day). The study of the point of action of its antimalarial activity in the erythrocytic cycle of Plasmodium falciparum demonstrated that it occurred at the mature trophozoite and young schizont stages. Moreover, ellagic acid has been shown to potentiate the activity of current antimalarial drugs such as chloroquine, mefloquine, artesunate, and atovaquone. This study also proved the antioxidant activity of ellagic acid and, in contrast, the inhibitory effect of the antioxidant compound N-acetyl-l-cysteine on its antimalarial efficacy. The possible mechanisms of action of ellagic acid on P. falciparum are discussed in light of the results. Ellagic acid has in vivo activity against plasmodia, but modification of the compound could lead to improved pharmacological properties, principally for the oral route.