Unlocking the potential of snake venom-based molecules against the malaria, Chagas disease, and leishmaniasis triad.

Malaria, leishmaniasis and Chagas disease are vector-borne protozoal infections with a disproportionately high impact on the most fragile societies in the world, and despite malaria-focused research gained momentum in the past two decades, both trypanosomiases and leishmaniases remain neglected tropical diseases. Affordable effective drugs remain the mainstay of tackling this burden, but toxicicty, inneficiency against later stage disease, and drug resistance issues are serious shortcomings. One strategy to overcome these hurdles is to get new therapeutics or inspiration in nature. Indeed, snake venoms have been recognized as valuable sources of biomacromolecules, like peptides and proteins, with antiprotozoal activity. This review highlights major snake venom components active against at least one of the three aforementioned diseases, which include phospholipases A2, metalloproteases, L-amino acid oxidases, lectins, and oligopeptides. The relevance of this repertoire of biomacromolecules and the bottlenecks in their clinical translation are discussed considering approaches that should increase the success rate in this arduous task. Overall, this review underlines how venom-derived biomacromolecules could lead to pioneering antiprotozoal treatments and how the drug landscape for neglected diseases may be revolutionized by a closer look at venoms. Further investigations on poorly studied venoms is needed and could add new therapeutics to the pipeline.

Meta-Analysis of MS-Based Proteomics Studies Indicates Interferon Regulatory Factor 4 and Nucleobindin1 as Potential Prognostic and Drug Resistance Biomarkers in Diffuse Large B Cell Lymphoma.

The prognosis of diffuse large B cell lymphoma (DLBCL) is inaccurately predicted using clinical features and immunohistochemistry (IHC) algorithms. Nomination of a panel of molecules as the target for therapy and predicting prognosis in DLBCL is challenging because of the divergences in the results of molecular studies. Mass spectrometry (MS)-based proteomics in the clinic represents an analytical tool with the potential to improve DLBCL diagnosis and prognosis. Previous proteomics studies using MS-based proteomics identified a wide range of proteins. To achieve a consensus, we reviewed MS-based proteomics studies and extracted the most consistently significantly dysregulated proteins. These proteins were then further explored by analyzing data from other omics fields. Among all significantly regulated proteins, interferon regulatory factor 4 (IRF4) was identified as a potential target by proteomics, genomics, and IHC. Moreover, annexinA5 (ANXA5) and nucleobindin1 (NUCB1) were two of the most up-regulated proteins identified in MS studies. Functional enrichment analysis identified the light zone reactions of the germinal center (LZ-GC) together with cytoskeleton locomotion functions as enriched based on consistent, significantly dysregulated proteins. In this study, we suggest IRF4 and NUCB1 proteins as potential biomarkers that deserve further investigation in the field of DLBCL sub-classification and prognosis.

Extracellular Vesicles in Diffuse Large B Cell Lymphoma: Characterization and Diagnostic Potential.

Diffuse large B cell lymphoma (DLBCL) is an aggressive B cell lymphoma characterized by a heterogeneous behavior and in need of more accurate biological characterization monitoring and prognostic tools. Extracellular vesicles are secreted by all cell types and are currently established to some extent as representatives of the cell of origin. The present study characterized and evaluated the diagnostic and prognostic potential of plasma extracellular vesicles (EVs) proteome in DLBCL by using state-of-the-art mass spectrometry. The EV proteome is strongly affected by DLBCL status, with multiple proteins uniquely identified in the plasma of DLBCL. A proof-of-concept classifier resulted in highly accurate classification with a sensitivity and specificity of 1 when tested on the holdout test data set. On the other hand, no proteins were identified to correlate with non-germinal center B-cell like (non-GCB) or GCB subtypes to a significant degree after correction for multiple testing. However, functional analysis suggested that antigen binding is regulated when comparing non-GCB and GCB. Survival analysis based on protein quantitative values and clinical parameters identified multiple EV proteins as significantly correlated to survival. In conclusion, the plasma extracellular vesicle proteome identifies DLBCL cancer patients from healthy donors and contains potential EV protein markers for prediction of survival.

Boosting Cosmeceutical Peptides: Coupling Imidazolium-Based Ionic Liquids to Pentapeptide-4 Originates New Leads with Antimicrobial and Collagenesis-Inducing Activities.

Following our previous reports on dual-action antibacterial and collagenesis-inducing hybrid peptide constructs based on "pentapeptide-4" (PP4, with amino acid sequence KTTKS), whose N-palmitoyl derivative is the well-known cosmeceutical ingredient Matrixyl, herein we disclose novel ionic liquid/PP4 conjugates (IL-KTTKS). These conjugates present potent activity against either antibiotic-susceptible strains or multidrug resistant clinical isolates of both Gram-positive and Gram-negative bacterial species belonging to the so-called "ESKAPE" group of pathogens. Noteworthy, their antibacterial activity is preserved in simulated wound fluid, which anticipates an effective action in the setting of a real wound bed. Moreover, their collagenesis-inducing effects in vitro are comparable to or stronger than those of Matrixyl. Altogether, IL-KTTKS exert a triple antibacterial, antifungal, and collagenesis-inducing action in vitro. These findings provide solid grounds for us to advance IL-KTTKS conjugates as promising leads for future development of topical treatments for complicated skin and soft tissue infections (cSSTI). Further studies are envisaged to incorporate IL-conjugates into suitable nanoformulations, to reduce toxicity and/or improve resistance to proteolytic degradation. IMPORTANCE As life expectancy increases, diseases causing chronic wound infections become more prevalent. Diabetes, peripheral vascular diseases, and bedridden patients are often associated with non-healing wounds that become infected, resulting in high morbidity and mortality. This is exacerbated by the fact that microbes are becoming increasingly resistant to antibiotics, so efforts must converge toward finding efficient therapeutic alternatives. Recently, our team identified a new type of constructs that combine (i) peptides used in cosmetics to promote collagen formation with (ii) imidazolium-based ionic liquids, which have antimicrobial and skin penetration properties. These constructs have potent wide-spectrum antimicrobial action, including against multidrug-resistant Gram-positive and Gram-negative bacteria, and fungi. Moreover, they can boost collagen formation. Hence, this is an unprecedented class of lead molecules toward development of a new topical medicine for chronically infected wounds.

Proteomic Landscape of Extracellular Vesicles for Diffuse Large B-Cell Lymphoma Subtyping.

The role of extracellular vesicles (EVs) proteome in diffuse large B-cell lymphoma (DLBCL) pathology, subclassification, and patient screening is unexplored. We analyzed by state-of-the-art mass spectrometry the whole cell and secreted extracellular vesicles (EVs) proteomes of different molecular subtypes of DLBCL, germinal center B cell (GCB subtype), and activated B cell (ABC subtype). After quality control assessment, we compared whole-cell and secreted EVs proteomes of the two cell-of-origin (COO) categories, GCB and ABC subtypes, resulting in 288/1115 significantly differential expressed proteins from the whole-cell proteome and 228/608 proteins from EVs (adjust p-value < 0.05/p-value < 0.05). In our preclinical model system, we demonstrated that the EV proteome and the whole-cell proteome possess the capacity to separate cell lines into ABC and GCB subtypes. KEGG functional analysis and GO enrichment analysis for cellular component, molecular function, and biological process of differential expressed proteins (DEP) between ABC and GCB EVs showed a significant enrichment of pathways involved in immune response function. Other enriched functional categories for DEPs constitute cellular signaling and intracellular trafficking such as B-cell receptor (BCR), Fc_gamma R-mediated phagocytosis, ErbB signaling, and endocytosis. Our results suggest EVs can be explored as a tool for patient diagnosis, follow-up, and disease monitoring. Finally, this study proposes novel drug targets based on highly expressed proteins, for which antitumor drugs are available suggesting potential combinatorial therapies for aggressive forms of DLBCL. Data are available via ProteomeXchange with identifier PXD028267.

On the Development of a Cutaneous Flavonoid Delivery System: Advances and Limitations.

Flavonoids are one of the vital classes of natural polyphenolic compounds abundantly found in plants. Due to their wide range of therapeutic properties, which include antioxidant, anti-inflammatory, photoprotective, and depigmentation effects, flavonoids have been demonstrated to be promising agents in the treatment of several skin disorders. However, their lipophilic nature and poor water solubility invariably lead to limited oral bioavailability. In addition, they are rapidly degraded and metabolized in the human body, hindering their potential contribution to the prevention and treatment of many disorders. Thus, to overcome these challenges, several cutaneous delivery systems have been extensively studied. Topical drug delivery besides offering an alternative administration route also ensures a sustained release of the active compound at the desired site of action. Incorporation into lipid or polymer-based nanoparticles appears to be a highly effective approach for cutaneous delivery of flavonoids with good encapsulation potential and reduced toxicity. This review focuses on currently available formulations used to administer either topically or systemically different classes of flavonoids in the skin, highlighting their potential application as therapeutic and preventive agents.

Evaluation of a worldwide EQA scheme for complex clonality analysis of clinical lymphoproliferative cases demonstrates a learning effect.

Clonality analysis of immunoglobulin (IG) or T-cell receptor (TR) gene rearrangements is routine practice to assist diagnosis of lymphoid malignancies. Participation in external quality assessment (EQA) aids laboratories in identifying systematic shortcomings. The aim of this study was to evaluate laboratories' improvement in IG/TR analysis and interpretation during five EQA rounds between 2014 and 2018. Each year, participants received a total of five cases for IG and five cases for TR testing. Paper-based cases were included for analysis of the final molecular conclusion that should be interpreted based on the integration of the individual PCR results. Wet cases were distributed for analysis of their routine protocol as well as evaluation of the final molecular conclusion. In total, 94.9% (506/533) of wet tests and 97.9% (829/847) of paper tests were correctly analyzed for IG, and 96.8% (507/524) wet tests and 93.2% (765/821) paper tests were correctly analyzed for TR. Analysis scores significantly improved when laboratories participated to more EQA rounds (p=0.001). Overall performance was significantly lower (p=0.008) for non-EuroClonality laboratories (95% for IG and 93% for TR) compared to EuroClonality laboratories (99% for IG and 97% for TR). The difference was not related to the EQA scheme year, anatomic origin of the sample, or final clinical diagnosis. This evaluation showed that repeated EQA participation helps to reduce performance differences between laboratories (EuroClonality versus non-EuroClonality) and between sample types (paper versus wet). The difficulties in interpreting oligoclonal cases highlighted the need for continued education by meetings and EQA schemes.

A new family of hydroxytyrosol phenolipids for the antioxidant protection of liposomal systems.

Hydroxytyrosol (HT) is a well-known olive oil polyphenol for its high antioxidant capacity and important cardio and neuroprotective effects. However, its use in lipidic systems is limited, due to its hydrophilic character. In this study, we approach the particular structure of xanthophylls and synthetize HT esters specially designed for the protection of liposomal systems. These HT esters contain two polyphenolic moieties separated by a lipophilic alkyl spacer of different length (12, 16 or 22 carbons). To evaluate the antioxidant activity of these compounds against the 2,2'-azobis(2-amidinopropane) hydrochloride induced oxidation, soybean phospholipid liposomes were used. Fluorescence quenching studies were used to assess the insertion of the compounds in the liposomes. The synthetized HT derivatives were able to protect liposomes from induced oxidation when added to the suspensions. The rank of activity was severely influenced by the alkyl chain length of the spacer molecule, being the C12 derivative the most active antioxidant, with an increase in the oxidative stability of liposomes of 2.2 times when compared with the control. The incorporation of compounds during liposome preparation improved the antioxidant capacity of all HT derivatives by about 2.8 times, when compared to the control. This is probably due to a similar transmembrane position with both polyphenolic rings located at the phospholipid polar heads. The synthesis of bis-ester derivatives seems to be a promising strategy to fine-tune antioxidant molecules at biomembranes, thus increasing the oxidative stability of liposomal systems by improving the antioxidant activity of hydrophilic phenolic compounds with high free radical scavenging activity.

Interaction of polyphenols with model membranes: Putative implications to mouthfeel perception.

Food polyphenols in fruits juices, tea, coffee, wine and beer confer sensory properties such as colour, astringency and bitterness. The development of functional healthy drinks without the unpleasant sensory feeling is boosting research for a clearer understanding on the interactions of polyphenols within the oral mucosa. In this study we investigated the interaction of astringent polyphenols, namely ECG, EGCG, procyanidin B4 and PGG, with lipids in model membranes by spectroscopic techniques. The membrane model was built varying the cholesterol content to mimic mouth regions and experiments were conducted at pH 5 to mimic the pH drop at the moment of beverage (e.g. green tea, red wine) intake. Fluorescence quenching results conducted on LUVs with cholesterol molar fractions ranging between 0.34 < χchol < 0.74 and similar size distributions (122.9 ± 3.7 nm) showed that interaction of polyphenols is structure- and concentration-dependent. Also, the decrease of partition constants (Kp) with increasing cholesterol content (χchol) suggest that the affinity of polyphenols is weaker in cholesterol-rich liposomes. STD results revealed that the interaction of EGCG and PGG with membrane lipids involved mainly galloyl residues. Overall, spectroscopic data show that polyphenols interact to higher extent with more polar regions found in buccal, flour of the mouth and gingiva regions than with more hydrophobic regions located in the palate and tongue supporting that lipid microenvironments play a role in oral sensory perception.

A New and Simple TRG Multiplex PCR Assay for Assessment of T-cell Clonality: A Comparative Study from the EuroClonality Consortium.

T-cell Receptor Gamma (TRG) rearrangements are commonly used to detect clonal lymphoproliferations in hematopathology, since they are rearranged in virtually all T lymphocytes and have a relatively limited recombinatorial repertoire, which reduces the risk of false negative results, at the cost of potential false positivity. We developed an initial one-tube, 2-fluorochrome EuroClonality TRG PCR multiplex (TRG-1T-2F) which was compared to the original 2-tube, 2-fluorochrome EuroClonality/BIOMED-2 TRG PCR (TRG-2T-2F) and a commercial Invivoscribe one-tube, one-fluorochrome kit (IVS-1T-1F) on a series of 239 samples, including both T-cell malignancies and reactive cases. This initial assay yielded discrepant results between the 10 participating EuroClonality laboratories when using 2 fluorochromes, leading to adoption of a final single color EuroClonality strategy (TRG-1T-1F). Compared to TRG-2T-2F, both TRG-1T-1F and IVS-1T-1F demonstrated easier interpretation and a lower risk of false positive from minor peaks in dispersed repertoires. Both generate smaller fragments and as such are likely to be better adapted to analysis of formalin-fixed paraffin-embedded (FFPE) tissue samples. Their differential performance was mainly explained by (i) superposition of biallelic rearrangements with IVS-1T-1F, due to more extensive overlapping of the repertoires and (ii) intentional omission of the TRGJP primer in TRG-1T-1F, in order to avoid the potential risk of confusion of consensus TRG V9-JP normal rearrangements with a pathological clone.

Turning a Collagenesis-Inducing Peptide Into a Potent Antibacterial and Antibiofilm Agent Against Multidrug-Resistant Gram-Negative Bacteria.

Antimicrobial resistance is becoming one the most serious health threats worldwide, as it not only hampers effective treatment of infectious diseases using current antibiotics, but also increases the risks of medical procedures like surgery, transplantation, bone and dental implantation, chemotherapy, or chronic wound management. To date, there are no effective measures to tackle life-threatening nosocomial infections caused by multidrug resistant bacterial species, of which Gram-negative species within the so-called "ESKAPE" pathogens are the most worrisome. Many such bacteria are frequently isolated from severely infected skin lesions such as diabetic foot ulcers (DFU). In this connection, we are pursuing new peptide constructs encompassing antimicrobial and collagenesis-inducing motifs, to tackle skin and soft tissue infections by exerting a dual effect: antimicrobial protection and faster healing of the wound. This produced peptide 3.1-PP4 showed MIC values as low as 1.0 and 2.1 µM against Escherichia coli and Pseudomonas aeruginosa, respectively, and low toxicity to HFF-1 human fibroblasts. Remarkably, the peptide was also potent against multidrug-resistant isolates of Klebsiella pneumoniae, E. coli, and P. aeruginosa (MIC values between 0.5 and 4.1 µM), and hampered the formation of/disaggregated K. pneumoniae biofilms of resistant clinical isolates. Moreover, this notable hybrid peptide retained the collagenesis-inducing behavior of the reference cosmeceutical peptide C16-PP4 ("Matrixyl"). In conclusion, 3.1-PP4 is a highly promising lead toward development of a topical treatment for severely infected skin injuries.

Assessment of sediment mutagenicity in areas under the influence of a contaminated site undergoing a remediation process.

Soil contamination enters aquatic ecosystems affecting sediment quality. The region studied is the Taquari River, Brazil, close to a site contaminated by wood preservatives, with a runoff route into the river. The first stage of the remediation process (In this article, the terms intervention and remediation have been used with slightly different meanings. We consider intervention to be the first phase of the remediation process, which aims to remove active sources) was an intervention to remove the main active sources. The Salmonella/microsome assay and polycyclic aromatic hydrocarbons (PAHs) were used to assess sediment quality in organic extracts during different intervention phases. The strains used were TA98, TA97a, and TA100 with and without S9mix (±S9). The results indicated the presence of pro-mutagens at site Ta010 (closest to the contaminated site) in all samplings, and the highest result occurred before intervention for TA100 + S9 (1,672 ± 215.9 rev/g). These values decreased during (83 ± 23.6 rev/g) and after this process (403 ± 105.9 rev/g), although the PAHs concentrations increased. Samples from this site presented PAHs with a carcinogenic potential during the assessed periods. After intervention, Ta006 (4 km downstream from Ta010) showed the most significant mutagenesis for TA100 + S9 (764 ± 230.2 rev/g) and, although the total PAHs values were lower, the species considered carcinogenic had higher concentrations. Mutagenesis predicted values of PAHs confirmed that carcinogenic species were predominantly detected by TA100, and the other PAHs by TA97a strains. Marked contaminant release to the river was observed, mainly in Ta010 at different periods. Mutagenicity and PAHs values in an internal stream, upstream from Ta010, showed a dispersion route of these agents. Thus, contamination in Ta010 and possible contribution to Ta006, after intervention, provides a warning regarding environmental quality in the region. Environ. Mol. Mutagen. 59:625-638, 2018. © 2018 Wiley Periodicals, Inc.

Monocarboxylate transporter 1 (MCT1), a tool to stratify acute myeloid leukemia (AML) patients and a vehicle to kill cancer cells.

Dysregulation of glucose/lactate dynamics plays a role in cancer progression, and MCTs are key elements in metabolic remodeling. VEGF is a relevant growth factor in the maintenance of bone marrow microenvironment and it is also important in hematological diseases. Our aim was to investigate the role of VEGF in the metabolic adaptation of Acute myeloid leukemia (AML) cells by evaluating the metabolic profiles and cell features according to the AML lineage and testing lactate as a metabolic coin. Our in vitro results showed that AML promyelocytic (HL60) and monocytic (THP1) (but not erythroid- HEL) lineages are well adapted to VEGF and lactate rich environment. Their metabolic adaptation relies on high rates of glycolysis to generate intermediates for PPP to support cell proliferation, and on the consumption of glycolysis-generated lactate to supply biomass and energy production. VEGF orchestrates this metabolic network by regulating MCT1 expression. Bromopyruvic acid (BPA) was proven to be an effective cytotoxic in AML, possibly transported by MCT1. Our study reinforces that targeting metabolism can be a good strategy to fight cancer. MCT1 expression at the time of diagnosis can assist on the identification of AML patients that will benefit from BPA therapy. Additionally, MCT1 can be used in targeted delivery of conventional cytotoxic drugs.

Thaulin-1: The first antimicrobial peptide isolated from the skin of a Patagonian frog Pleurodema thaul (Anura: Leptodactylidae: Leiuperinae) with activity against Escherichia coli.

Patagonia's biodiversity has been explored from many points of view, however, skin secretions of native amphibians have not been evaluated for antimicrobial peptide research until now. In this sense, Pleurodema thaul is the first amphibian specie to be studied from this large region of South America. Analysis of cDNA-encoding peptide in skin samples allowed identification of four new antimicrobial peptides. The predicted mature peptides were synthesized and all of them showed weak or null antimicrobial activity against Klebsiella pneumoniae, Staphylococcus aureus and Escherichia coli with the exception of thaulin-1, a cationic 26-residue linear, amphipathic, Gly- and Leu-rich peptide with moderate antimicrobial activity against E. coli (MIC of 24.7µM). AFM and SPR studies suggested a preferential interaction between these peptides and bacterial membranes. Cytotoxicity assays showed that thaulin peptides had minimal effects at MIC concentrations towards human and animal cells. These are the first peptides described for amphibians of the Pleurodema genus. These findings highlight the potential of the Patagonian region's unexplored biodiversity as a source for new molecule discovery.

Bisphenol A migration from plastic materials: direct insight of ecotoxicity in Daphnia magna.

Bisphenol A (BPA) is an endocrine disrupting chemical (EDC) whose migration from food packaging is recognized worldwide. However, the real overall food contamination and related consequences are yet largely unknown. Among humans, children's exposure to BPA has been emphasized because of the immaturity of their biological systems. The main aim of this study was to assess the reproductive impact of BPA leached from commercially available plastic containers used or related to child nutrition, performing ecotoxicological tests using the biomonitoring species Daphnia magna. Acute and chronic tests, as well as single and multigenerational tests were done. Migration of BPA from several baby bottles and other plastic containers evaluated by GC-MS indicated that a broader range of foodstuff may be contaminated when packed in plastics. Ecotoxicological test results performed using defined concentrations of BPA were in agreement with literature, although a precocious maturity of daphnids was detected at 3.0 mg/L. Curiously, an increased reproductive output (neonates per female) was observed when daphnids were bred in the polycarbonate (PC) containers (145.1 ± 4.3 % to 264.7 ± 3.8 %), both in single as in multigenerational tests, in comparison with the negative control group (100.3 ± 1.6 %). A strong correlated dose-dependent ecotoxicological effect was observed, providing evidence that BPA leached from plastic food packaging materials act as functional estrogen in vivo at very low concentrations. In contrast, neonate production by daphnids cultured in polypropylene and non-PC bottles was slightly but not significantly enhanced (92.5 ± 2.0 % to 118.8 ± 1.8 %). Multigenerational tests also revealed magnification of the adverse effects, not only on fecundity but also on mortality, which represents a worrying trend for organisms that are chronically exposed to xenoestrogens for many generations. Two plausible explanations for the observed results could be given: a non-monotonic dose-response relationship or a mixture toxicity effect.

On CK2 regulation of chronic lymphocytic leukemia cell viability.

Specific inhibition of signaling elements essential for the viability of B-cell chronic lymphocytic leukemia (CLL) cells offers great promise for the design of more efficient therapies. The protein serine/threonine kinase CK2 is frequently upregulated in cancer, and it is overexpressed and hyperactivated in primary CLL cells from untreated patients. We have shown that inhibition of CK2 induces apoptosis of CLL cells, whereas it does not significantly impact normal lymphocytes, demonstrating the selectivity of the CK2 inhibitors toward leukemia cells. Notably, although co-culture with OP9 stromal cells and BCR stimulation both promote leukemia cell survival in vitro, they do not prevent apoptosis of CLL cells treated with CK2 inhibitors. PI3K signaling pathway was previously shown to be essential for CLL cell viability, an observation we confirmed in all patient samples analyzed. Further, we observed that CK2 blockade decreases PTEN phosphorylation, leading to PTEN activation, and that apoptosis of CLL cells upon CK2 inhibition is mediated by PKC inactivation. This suggests that activation of PI3K/PKC signaling pathway is involved in the pro-survival effects of CK2 in CLL cells. Sensitivity to CK2 inhibition does not correlate with expression of ZAP-70 or CD38, or with IGVH mutation status. However, it positively correlates with the percentage of CLL cells in the peripheral blood, ß2 microglobulin levels, and Binet clinical stage. CK2 appears to play an important role in the biology of CLL and constitutes a promising target for the development of leukemia-specific therapies.

Targeting CK2 overexpression and hyperactivation as a novel therapeutic tool in chronic lymphocytic leukemia.

Expression of protein kinase CK2 is frequently deregulated in cancer and mounting evidence implicates CK2 in tumorigenesis. Here, we show that CK2 is overexpressed and hyperactivated in chronic lymphocytic leukemia (CLL). Inhibition of CK2 induces apoptosis of CLL cells without significantly affecting normal B and T lymphocytes. Importantly, this effect is not reversed by coculture with OP9 stromal cells, which are otherwise capable of rescuing CLL cells from in vitro spontaneous apoptosis. CLL cell death upon CK2 inhibition is mediated by inactivation of PKC, a PI3K downstream target, and correlates with increased PTEN activity, indicating that CK2 promotes CLL cell survival at least in part via PI3K-dependent signaling. Although CK2 antagonists induce significant apoptosis of CLL cells in all patient samples analyzed, sensitivity to CK2 blockade positively correlates with the percentage of CLL cells in the peripheral blood, ß2 microglobulin serum levels and clinical stage. These data suggest that subsets of patients with aggressive and advanced stage disease may especially benefit from therapeutic strategies targeting CK2 function. Overall, our study indicates that CK2 plays a critical role in CLL cell survival, laying the groundwork for the inclusion of CK2 inhibitors into future therapeutic strategies.

Solution and biological behaviour of enrofloxacin metalloantibiotics: a route to counteract bacterial resistance?

Solution behaviour of enrofloxacin complexes with copper(II), nickel(II), cobalt(II) and zinc(II) in the presence and absence of 1, 10-phenanthroline was studied in aqueous solution, by potentiometry. The results obtained show that under physiological conditions (micromolar concentration range and pH 7.4) only copper(II) forms stable complexes. Binary copper(II)/enrofloxacin and ternary copper(II)/enrofloxacin/phenanthroline complexes were synthesised and characterized by elemental analysis, UV-visible spectroscopy and FTIR. The antimicrobial activity of these complexes and of copper(II)/enrofloxacin and copper(II)/enrofloxacin/phenanthroline solutions, prepared by mixing of the individual components in the same stoichiometric proportion and concentration range used for the synthesised complexes, was tested against two different Escherichia coli strains. Although, at a glance, the results point to a possible use of both complexes as metalloantibiotics, a detailed analysis shows that, at biological concentrations, the copper(II) binary complex does not exist and the antimicrobial activity observed is a consequence of its dissociation into free enrofloxacin. Consequently, only the ternary complex seems worth pursuing as a possible antimicrobial agent candidate. Moreover, as the biological studies showed, both the synthesised complexes and the solutions prepared by mixing the components exhibited the same behaviour. Hence, a new, faster and accurate methodology to screen metalloantibiotics prior to synthesis of the complexes is proposed.

Fluorescent 3-hydroxy-4-pyridinone hexadentate iron chelators: intracellular distribution and the relevance to antimycobacterial properties.

We report the synthesis and characterization of a fluorescent iron chelator (4), shown to be effective in inhibiting the growth of Mycobacterium avium in macrophages, together with the synthesis and characterization of two unsuccessful analogues selected to facilitate identification of the molecular properties responsible for the antimicrobial activity. Partition of the chelators in liposomes was investigated and the compounds were assessed with respect to uptake by macrophages, responsiveness to iron overload/iron deprivation and intracellular distribution by flow cytometry and confocal microscopy. The synthesis of the hexadentate chelators is based on a tetrahedral structure to which three bidentate 3-hydroxy-4-pyridinone chelating units are linked via amide bonds. The structure is synthetically versatile, allowing further addition of functional groups such as fluorophores. Here, we analyse the non-functionalized hexadentate unit (3) and the corresponding rhodamine B (4) and fluorescein (5) labelled chelators. The iron(III) stability constant was determined for 3 and the values log beta = 34.4 and pFe(3+) = 29.8 indicate an affinity for iron of the same order of magnitude as that of mycobacteria siderophores. Fluorescence properties in the presence of liposomes show that 4 strongly interacts with the lipid phase, whereas 5 does not. Such different behaviour may explain their distinct intracellular localization as revealed by confocal microscopy. The flow cytometry and confocal microscopy studies indicate that 4 is readily engulfed by macrophages and targeted to cytosol and vesicles of the endolysosomal continuum, whereas 5 is differentially distributed and only partially colocalizes with 4 after prolonged incubation. Differential distribution of the compounds is likely to account for their different efficacy against mycobacteria.

Benzodiazepine-mediated structural changes in the multidrug transporter P-glycoprotein: an intrinsic fluorescence quenching analysis.

P-glycoprotein expressed in Pichia pastoris was used to study the drug binding sites of different benzodiazepines. The effect of bromazepam, chlordiazepoxide, diazepam and flurazepam on P-glycoprotein structure was investigated by measuring the intrinsic fluorescence of the transporter tryptophan residues. Purified mouse mdr1a transporter in mixed micelles of 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid and 1,2-dimiristoyl-sn-glycerol-3-phosphocholine emitted fluorescence at 340 nm indicative of the fluorophores in a relatively apolar environment. Acrylamide and iodide ion were used as collisional quenchers toward distinct regions of the transporter, the protein and the interface protein-surface, respectively. Binding of ATP induced conformational changes at the protein surface level in accordance with the location of the nucleotide binding sites. Bromazepam interaction with the transporter was located at the protein-surface interface, diazepam at the membrane region and chlordiazepoxide at the protein surface. Only the flurazepam interaction site was not detected by the quenchers used. All benzodiazepines were able to elicit reorientation of the protein fluorophores on the P-glycoprotein-ATP complex.