Molecular mechanism of glutaminase activation through filamentation and the role of filaments in mitophagy protection.

Glutaminase (GLS), which deaminates glutamine to form glutamate, is a mitochondrial tetrameric protein complex. Although inorganic phosphate (Pi) is known to promote GLS filamentation and activation, the molecular basis of this mechanism is unknown. Here we aimed to determine the molecular mechanism of Pi-induced mouse GLS filamentation and its impact on mitochondrial physiology. Single-particle cryogenic electron microscopy revealed an allosteric mechanism in which Pi binding at the tetramer interface and the activation loop is coupled to direct nucleophile activation at the active site. The active conformation is prone to enzyme filamentation. Notably, human GLS filaments form inside tubulated mitochondria following glutamine withdrawal, as shown by in situ cryo-electron tomography of cells thinned by cryo-focused ion beam milling. Mitochondria with GLS filaments exhibit increased protection from mitophagy. We reveal roles of filamentous GLS in mitochondrial morphology and recycling.

Toxicity and outcome of adults with acute myeloid leukemia receiving consolidation with high-dose cytarabine.

INTRODUCTION: High-dose cytarabine is considered standard of care as consolidation chemotherapy in adults with acute myeloid leukemia (AML) who are not eligible for allogeneic hematopoietic cell transplantation, but may be associated with significant toxicity. We evaluated the toxicity associated with high-dose cytarabine given as consolidation in AML patients treated at a Brazilian public hospital. METHODS: We retrospectively reviewed the charts of all patients with AML treated between 2008 and 2020 who obtained complete remission (CR) after one cycle of induction chemotherapy and received consolidation with at least one cycle of high-dose cytarabine (defined as 3 g/m2 every 12 h days 1, 3 and 5). RESULTS: Among 61 patients who received induction remission, 32 obtained CR and 28 received at least one cycle of high-dose cytarabine, for a total of 67 cycles (median 2 cycles per patient, range 1 - 4). In 45 cycles (67.2%) the patient was discharged after the end of chemotherapy, with a median of 6 days at home (range 3 - 8). Readmission occurred in 31 of the 45 cycles (68.9%). The most frequent toxicities were febrile neutropenia (56.7%), nausea and vomiting (23.9%), oral mucositis (14.9%) and diarrhea (11.9%). Bacteremia was documented in 13 cycles (34.2%). There were three cases of typhlitis and two of invasive fungal disease (aspergillosis and candidemia). Four patients died (14.3%), with two deaths considered treatment-related (candidemia and typhlitis). CONCLUSION: In the setting of a Brazilian public hospital, high-dose cytarabine as consolidation therapy is feasible, with manageable toxicity profile.

Mechanism of high-mannose N-glycan breakdown and metabolism by Bifidobacterium longum.

Bifidobacteria are early colonizers of the human gut and play central roles in human health and metabolism. To thrive in this competitive niche, these bacteria evolved the capacity to use complex carbohydrates, including mammalian N-glycans. Herein, we elucidated pivotal biochemical steps involved in high-mannose N-glycan utilization by Bifidobacterium longum. After N-glycan release by an endo-ß-N-acetylglucosaminidase, the mannosyl arms are trimmed by the cooperative action of three functionally distinct glycoside hydrolase 38 (GH38) α-mannosidases and a specific GH125 α-1,6-mannosidase. High-resolution cryo-electron microscopy structures revealed that bifidobacterial GH38 α-mannosidases form homotetramers, with the N-terminal jelly roll domain contributing to substrate selectivity. Additionally, an α-glucosidase enables the processing of monoglucosylated N-glycans. Notably, the main degradation product, mannose, is isomerized into fructose before phosphorylation, an unconventional metabolic route connecting it to the bifid shunt pathway. These findings shed light on key molecular mechanisms used by bifidobacteria to use high-mannose N-glycans, a perennial carbon and energy source in the intestinal lumen.

PEGylated versus Non-PEGylated pH-Sensitive Liposomes: New Insights from a Comparative Antitumor Activity Study.

PEGylated liposomes are largely studied as long-circulating drug delivery systems. Nevertheless, the addition of PEG can result in reduced interactions between liposomes and cells, hindering liposomal internalization into target cells. The presence of PEG on the surface of pH-sensitive liposomes is not advantageous in terms of biodistribution and tumor uptake, raising the question of whether the indiscriminate use of PEG benefits the formulation. In this study, two doxorubicin-loaded pH-sensitive liposomal formulations, PEGylated (Lip2000-DOX) or non-PEGylated (Lip-DOX), were prepared and characterized. Overall, the PEGylated and non-PEGylated liposomes showed no differences in size or morphology in Cryo-TEM image analysis. Specifically, DLS analysis showed a mean diameter of 140 nm, PDI lower than 0.2, and zeta potential close to neutrality. Both formulations showed an EP higher than 90%. With respect to drug delivery, Lip-DOX had better cellular uptake than Lip2000-DOX, suggesting that the presence of PEG reduced the amount of intracellular DOX accumulation. The antitumor activities of free-DOX and both liposomal formulations were evaluated in 4T1 breast tumor-bearing BALB/c mice. The results showed that Lip-DOX was more effective in controlling tumor growth than other groups, inhibiting tumor growth by 60.4%. Histological lung analysis confirmed that none of the animals in the Lip-DOX group had metastatic foci. These results support that pH-sensitive liposomes have interesting antitumor properties and may produce important outcomes without PEG.

An atomic model for the human septin hexamer by cryo-EM.

In order to form functional filaments, human septins must assemble into hetero-oligomeric rod-like particles which polymerize end-to-end. The rules governing the assembly of these particles and the subsequent filaments are incompletely understood. Although crystallographic approaches have been successful in studying the separate components of the system, there has been difficulty in obtaining high resolution structures of the full particle. Here we report a first cryo-EM structure for a hexameric rod composed of human septins 2, 6 and 7 with a global resolution of ~3.6 Å and a local resolution of between ~3.0 Å and ~5.0 Å. By fitting the previously determined high-resolution crystal structures of the component subunits into the cryo-EM map, we are able to provide an essentially complete model for the particle. This exposes SEPT2 NC-interfaces at the termini of the hexamer and leaves internal cavities between the SEPT6-SEPT7 pairs. The floor of the cavity is formed by the two α0 helices including their polybasic regions. These are locked into place between the two subunits by interactions made with the α5 and α6 helices of the neighbouring monomer together with its polyacidic region. The cavity may serve to provide space allowing the subunits to move with respect to one another. The elongated particle shows a tendency to bend at its centre where two copies of SEPT7 form a homodimeric G-interface. Such bending is almost certainly related to the ability of septin filaments to recognize and even induce membrane curvature.

Cryo-EM structure of the mature and infective Mayaro virus at 4.4 Å resolution reveals features of arthritogenic alphaviruses.

Mayaro virus (MAYV) is an emerging arbovirus of the Americas that may cause a debilitating arthritogenic disease. The biology of MAYV is not fully understood and largely inferred from related arthritogenic alphaviruses. Here, we present the structure of MAYV at 4.4 Å resolution, obtained from a preparation of mature, infective virions. MAYV presents typical alphavirus features and organization. Interactions between viral proteins that lead to particle formation are described together with a hydrophobic pocket formed between E1 and E2 spike proteins and conformational epitopes specific of MAYV. We also describe MAYV glycosylation residues in E1 and E2 that may affect MXRA8 host receptor binding, and a molecular "handshake" between MAYV spikes formed by N262 glycosylation in adjacent E2 proteins. The structure of MAYV is suggestive of structural and functional complexity among alphaviruses, which may be targeted for specificity or antiviral activity.

Structure optimization of lipopeptide assemblies for aldol reactions in an aqueous medium.

Four amphiphilic peptides were synthesized, characterized, and evaluated regarding their efficiency in the catalysis of direct aldol reactions in water. The lipopeptides differ by having a double lipid chain and a guanidinium pyrrole group functionalizing one Lys side chain. All the samples are composed of the amino acids l-proline (P), l-arginine (R), or l-lysine (K) functionalized with the cationic guanidiniocarbonyl pyrrole unit (GCP), l-tryptophan (W), and l-glycine (G), covalently linked to one or two long aliphatic chains, leading to surfactant-like designs with controlled proline protonation state and different stereoselectivity. Critical aggregation concentrations (cac) were higher in the presence of the GCP group, suggesting that self-assembly depends on charge distribution along the peptide backbone. Cryogenic Transmission Electron Microscopy (Cryo-TEM) and Small Angle X-ray Scattering (SAXS) showed a rich polymorphism including spherical, cylindrical, and bilayer structures. Molecular dynamics simulations performed to assess the lipopeptide polymorphs revealed an excellent agreement with core-shell arrangements derived from SAXS data and provided an atomistic view of the changes incurred by modifying head groups and lipid chains. The resulting nanostructures behaved as excellent catalysts for aldol condensation reactions, in which superior conversions (>99%), high diastereoselectivities (ds = 94 : 6), and enantioselectivities (ee = 92%) were obtained. Our findings contribute to elucidate the effect of nanoscale organization of lipopeptide assemblies in the catalysis of aldol reactions in an aqueous environment.

Current treatment preferences in acute myeloid leukemia: a survey in Brazil

ABSTRACT Introduction:: Most adults with acute myeloid leukemia (AML) will eventually relapse from their disease. The combination of 7-day cytarabine and an anthracycline on days 1-3 (the so called "7 + 3" regimen) can be considered standard of care of younger patients with AML. However, the treatment of the elderly ineligible for intensive chemotherapy remains a challenge. Low-dose of subcutaneous cytarabine or hypomethylating agents (HMA) have been studied this group. There are no studies investigating physician practice variation in treating AML in Brazil. Methods:: We developed a survey with ten questions in order to explore the approach to AML in Brazil. Results:: The sample size comprised 100 hematologists. Most reported regular (63%) or occasional (29%) treatment of AML patients. Karyotype analysis and polymerase chain reaction were available in 88% and 71% of institutions, respectively. Next generation sequencing analysis was used in 7% of instituitions. Younger patients receive the "7 + 3" protocol with continuous infusion of cytarabine and anthracycline in 98% of cases. The preferred anthracycline is daunorubicin (64%), followed by idarubicin (34%). The most prescribed daunorubicin dose was 60 mg/m2 (56%). Consolidation after CR with high cytarabine doses (HIDAC) was indicated by 84% of hematologists and 70% use 3 g/m2 twice a day for 3 days. Elderly and unfit patients received HMA (47%) as the preferred treatment. Conclusion:: We showed that the most prevalent AML treatments were according to current guidelines. There is room to improve on the availability of diagnostic tools and the capacity to perform bone marrow transplantation.

Bacterioruberin from Haloarchaea plus dexamethasone in ultra-small macrophage-targeted nanoparticles as potential intestinal repairing agent.

Oral administration of antioxidant and anti-inflammatory drugs have the potential to improve the current therapy of inflammatory bowel disease. Success of oral treatments, however, depends on the capacity of drugs to remain structurally stable along the gastrointestinal tract, and on the feasibility of accessing the target cells. Delivering anti-inflammatory and antioxidant drugs to macrophages using targeted nanoparticles, could make treatments more efficient. In this work structural features and in vitro activity of macrophage-targeted nanostructured archaeolipid carriers (NAC) containing the high antioxidant dipolar C50 carotenoid bacterioruberin (BR) plus dexamethasone (Dex): NAC-Dex, are described. Ultra-small (66 nm), -32 mV ζ potential, 1200 µg Dex /ml NAC-Dex, consisted of a compritol and BR core, covered by a shell of sn 2,3 ether linked archaeolipids and Tween 80 (2: 2: 1.2: 3 % w/w) were obtained. NAC-Dex were extensively captured by macrophages and Caco-2 cells and displayed high anti-inflammatory and antioxidant activities on a gut inflammation model made of Caco-2 cells and lipopolysaccharide stimulated THP-1 derived macrophages reducing 65 % and 55 % TNF-α and IL-8 release, respectively and 60 % reactive oxygen species production. NAC-Dex also reversed the morphological changes induced by inflammation and increased the transepithelial electrical resistance, partly reconstituting the barrier function. Activity of BR and Dex in NAC-Dex was partially protected after simulated gastrointestinal digestion, improving the chances of BR-Dex joint activity. Results suggest that oral NAC-Dex deserve further exploration as intestinal barrier repairing agent.

Pair Distribution Function from Electron Diffraction in Cryogenic Electron Microscopy: Revealing Glassy Water Structure.

In recent years, cryogenic electron microscopy (Cryo-EM) has revolutionized the structure determination of wet samples and especially that of biological macromolecules. The glassy-water medium in which the molecules are embedded is considered an almost in vivo environment for biological samples. The local structure of amorphous ice is known from neutron- and X-ray-diffraction studies, techniques appropriate for much larger volumes than those used in cryo-EM. We here present a first study of the pair-distribution function g(r) of glassy water under cryo-EM conditions using electron diffraction data. We found g(r) to be between that of low-density amorphous ice and that of supercooled water. Under electron exposure, cubic-ice regions were found to nucleate in thicker glassy-water samples. Our work enables to obtain quantitative structural information using g(r) from cryo-EM.

Nanoparticles containing ß-cyclodextrin potentially useful for the treatment of Niemann-Pick C.

ß-Cyclodextrin (ß-CD) is being considered a promising therapy for Niemann-Pick C (NPC) disease because of its ability to mobilise the entrapped cholesterol from lysosomes, however, a major limitation is its inability to cross the blood-brain barrier (BBB) and address the central nervous system (CNS) manifestations of the disease. Considering this, we aimed to design nanoparticles able to cross the BBB and deliver ß-CD into the CNS lysosomes. The physicochemical characteristics of ß-CD-loaded nanoparticles were evaluated by dynamic light scattering, small-angle X-ray scattering, and cryogenic transmission electron microscopy. The in vitro analyses were performed with NPC dermal fibroblasts and the ß-CD-loaded nanoparticles were tracked in vivo. The nanoparticles showed a mean diameter around 120 nm with a disordered bicontinuous inner structure. The nanoparticles did not cause decrease in cell viability, impairment in the antioxidant enzymes activity, damage to biomolecules or release of reactive species in NPC dermal fibroblasts; also, they did not induce genotoxicity or alter the mitochondrial function in healthy fibroblasts. The ß-CD-loaded nanoparticles were taken up by lysosomes reducing the cholesterol accumulated in NPC fibroblasts and reached the CNS of mice more intensely than other organs, demonstrating advantages compared to the free ß-CD. The results demonstrated the potential of the ß-CD-loaded nanoparticles in reducing the brain impairment of NPC.

Current treatment preferences in acute myeloid leukemia: a survey in Brazil.

INTRODUCTION: Most adults with acute myeloid leukemia (AML) will eventually relapse from their disease. The combination of 7-day cytarabine and an anthracycline on days 1-3 (the so called "7 + 3" regimen) can be considered standard of care of younger patients with AML. However, the treatment of the elderly ineligible for intensive chemotherapy remains a challenge. Low-dose of subcutaneous cytarabine or hypomethylating agents (HMA) have been studied this group. There are no studies investigating physician practice variation in treating AML in Brazil. METHODS: We developed a survey with ten questions in order to explore the approach to AML in Brazil. RESULTS: The sample size comprised 100 hematologists. Most reported regular (63%) or occasional (29%) treatment of AML patients. Karyotype analysis and polymerase chain reaction were available in 88% and 71% of institutions, respectively. Next generation sequencing analysis was used in 7% of instituitions. Younger patients receive the "7 + 3" protocol with continuous infusion of cytarabine and anthracycline in 98% of cases. The preferred anthracycline is daunorubicin (64%), followed by idarubicin (34%). The most prescribed daunorubicin dose was 60 mg/m2 (56%). Consolidation after CR with high cytarabine doses (HIDAC) was indicated by 84% of hematologists and 70% use 3 g/m2 twice a day for 3 days. Elderly and unfit patients received HMA (47%) as the preferred treatment. CONCLUSION: We showed that the most prevalent AML treatments were according to current guidelines. There is room to improve on the availability of diagnostic tools and the capacity to perform bone marrow transplantation.

Characterization of phospholipid vesicles containing lauric acid: physicochemical basis for process and product development.

Lauric acid (LAH) strongly inhibits the growth of acne-causing bacteria. LAH is essentially water-insoluble and the solubility of laurate (LA) salts are medium and temperature dependent. Hence, LAH/LA preparations are difficult to formulate. Here we fully characterized phospholipid vesicles containing up to 50 mol% LAH. Vesicles of dipalmitoylphosphatidylcholine (DPPC) containing LAH, at pHs 7.4 and 5.0, were characterized measuring size, charge, bilayer phase transition temperature (Tm) and permeability of water-soluble probes. Small angle X-ray scattering and cryotransmission electron microscopy showed multilamellar vesicles at low LAH %. Increasing LAH % had a negligible effect on particle size. An internal aqueous compartment in all vesicle's preparations, even at equimolar DPPC: LAH fractions, was demonstrated using water-soluble probes. At pH 5.0, the interaction between DPPC and LAH increased the Tm and phase transition cooperativity showing a single lipid phase formed by hydrogen-bonded DPPC: LAH complexes. At pH 7.4, vesicles containing 50 mol% LAH exhibited distinct phases, ascribed to complex formation between LAH and LA or LAH and DPPC. LAH incorporated in the vesicles minimally permeated a skin preparation at both pHs, indicating that the primary sites of LAH solubilization were the skin layers. These results provide the foundations for developing processes and products containing DPPC: LAH.

A revised order of subunits in mammalian septin complexes.

Septins are GTP binding proteins considered to be novel components of the cytoskeleton. They polymerize into filaments based on hexameric or octameric core particles in which two copies of either three or four different septins, respectively, assemble into a specific sequence. Viable combinations of the 13 human septins are believed to obey substitution rules in which the different septins involved must come from distinct subgroups. The hexameric assembly, for example, has been reported to be SEPT7-SEPT6-SEPT2-SEPT2-SEPT6-SEPT7. Here, we have replaced SEPT2 by SEPT5 according to the substitution rules and used transmission electron microscopy to demonstrate that the resulting recombinant complex assembles into hexameric particles which are inverted with respect that predicted previously. MBP-SEPT5 constructs and immunostaining show that SEPT5 occupies the terminal positions of the hexamer. We further show that this is also true for the assembly including SEPT2, in direct contradiction with that reported previously. Consequently, both complexes expose an NC interface, as reported for yeast, which we show to be more susceptible to high salt concentrations. The correct assembly for the canonical combination of septins 2-6-7 is therefore established to be SEPT2-SEPT6-SEPT7-SEPT7-SEPT6-SEPT2, implying the need for revision of the mechanisms involved in filament assembly.

Determination of fluorescein and brominated fluoresceins in the color additive D&C Orange No. 5 and its lakes using high-performance liquid chromatography.

The colour additives D&C Orange No. 5 (O5) and its lakes (O5L) are subject to batch certification by the U.S. Food and Drug Administration (FDA) to ensure compliance with specifications in the Code of Federal Regulations (CFR). The present study reports the development of a high-performance liquid chromatography (HPLC) method for the quantitative determination of seven CFR-specified components in O5 and O5L - fluorescein and six brominated fluoresceins. The analytes were quantified using six-point calibration curves with data points (w/w) that ranged as follows: 20.0-70.0% for 4',5'-dibromofluorescein; 9.8-44.1% for 2',4',5'-tribromofluorescein; 1.01-15.2% for 2',4',5',7'-tetrabromofluorescein; 0.10-3.12% for 2',4'-dibromofluorescein; 0.10-3.06% for 2',5'-dibromofluorescein; 0.11-2.85% for 4'-bromofluorescein; and 0.10-2.02% for fluorescein. For all seven analytes, the HPLC instrument response was linear (R2 > 0.999) over the tested concentration ranges and the limits of detection (0.01-1.55%) were well below the CFR-specified levels. Other validation data showed good analyte recovery (87.91-101.73%) as well as method precision measured by the relative standard deviation (0.53-1.56%). The new method was applied to the analysis of test portions from 15 batches of O5 and eight batches of O5L submitted to FDA for certification by domestic and foreign manufacturers. Compared to the thin-layer chromatography/spectrophotometric procedure currently used for routine batch-certification analyses, the new method was found to be more sensitive, simpler to implement, and significantly faster, requiring 25 minutes rather than six hours to analyse one sample.

Interaction of graphene oxide with cell culture medium: Evaluating the fetal bovine serum protein corona formation towards in vitro nanotoxicity assessment and nanobiointeractions.

The interaction of single-layer graphene oxide (SLGO) and multi-layered graphene oxide (MLGO) with a cell culture medium (i.e. DMEM) was studied by evaluating fetal bovine serum (FBS) protein corona formation towards in vitro nanotoxicity assessment and nanobiointeractions. SLGO and MLGO exhibited different colloidal behavior in the culture medium, which was visualized by cryogenic transmission electron microscopy in situ analysis. Exploring proteomics and bioinformatics tools, 394 and 290 proteins were identified on the SLGO and MLGO hard corona compositions, respectively. From this amount, 115 proteins were exclusively detected on the SLGO and merely 11 on MLGO. SLGO enriched FBS proteins involved in metabolic processes and signal transduction, while MLGO enriched proteins involved in cellular development/structure, and lipid transport/metabolic processes. Such a distinct corona profile is due to differences on surface chemistry, aggregation behavior and the surface area of GO materials. Hydrophilic interactions were found to play a greater role in protein adsorption by MLGO than SLGO. Our results point out implications for in vitro studies of graphene oxide materials concerning the effective dose delivered to cells and corona bioactivity. Finally, we demonstrated the importance of integrating conventional and modern techniques thoroughly to understand the GO-FBS complexes towards more precise, reliable and advanced in vitro nanotoxicity assessment.

Superoxide dismutase in nanoarchaeosomes for targeted delivery to inflammatory macrophages.

Oxidative stress plays an essential role in the pathogenesis and progression of inflammatory bowel disease. Co-administration of antioxidants and anti-inflammatory drugs has shown clinical benefits. Due to its significant reactive oxygen species (ROS) scavenging ability, great interest has been focused on superoxide dismutase (SOD) for therapeutic use. However, oral SOD is exposed to biochemical degradation along gastrointestinal transit. Furthermore, the antioxidant activity of SOD must be achieved intracellularly, therefore its cell entry requires endocytic mediating mechanisms. In this work, SOD was loaded into nanoarchaeosomes (ARC-SOD), nanovesicles fully made of sn 2,3 ether linked phytanyl saturated archaeolipids to protect and target SOD to inflammatory macrophages upon oral administration. Antioxidant and anti-inflammatory activities of ARC-SOD, non-digested and digested in simulated gastrointestinal fluids, on macrophages stimulated with H2O2 and lipopolysaccharide were determined and compared with those of free SOD and SOD encapsulated into highly stable liposomes (LIPO-SOD). Compared to SOD and LIPO-SOD, ARC-SOD (170 ± 14 nm, -30 ± 4 mV zeta potential, 122 mg protein/g phospholipids) showed the highest antioxidant and anti-inflammatory activity: it reversed the cytotoxic effect of H2O2, decreased intracellular ROS and completely suppressed the production of IL-6 and TNF-α on stimulated J774 A.1 cells. Moreover, while the activity of LIPO-SOD was lost upon preparation, gastrointestinal digestion and storage, ARC-SOD was easy to prepare and retained its antioxidant capacity upon digestion in simulated gastrointestinal fluids and after 5 months of storage. Because of their structural and pharmacodynamic features, ARC-SOD may be suitable for oral targeted delivery of SOD to inflamed mucosa.

The anti MRSA biofilm activity of Thymus vulgaris essential oil in nanovesicles.

BACKGROUND: Thymus vulgaris essential oil (T) could be an alternative to classical antibiotics against bacterial biofilms, which show increased tolerance to antibiotics and host defence systems and contribute to the persistence of chronic bacterial infections. HYPOTHESIS: A nanovesicular formulation of T may chemically protect the structure and relative composition of its multiple components, potentially improving its antibacterial and antibiofilm activity. STUDY DESIGN: We prepared and structurally characterized T in two types of nanovesicles: nanoliposomes (L80-T) made of Soybean phosphatidylcholine (SPC) and Polysorbate 80 (P80) [SPC:P80:T 1:0.75:0.3 w:w], and nanoarchaeosomes (A80-T) made of SPC, P80 and total polar archaeolipids (TPA) extracted from archaebacteria Halorubrum tebenquichense [SPC:TPA:P80:T 0.5:0.50.75:0.7 w:w]. We determined the macrophage cytotoxicity and the antibacterial activity against Staphylococcus aureus ATCC 25,923 and four MRSA clinical strains. RESULTS: L80-T (Z potential -4.1 ±â€¯0.6 mV, ∼ 115 nm, ∼ 22 mg/ml T) and A80-T (Z potential -6.6 ±â€¯1.5 mV, ∼ 130 nm, ∼ 42 mg/ml T) were colloidally and chemically stable, maintaining size, PDI, Z potential and T concentration for at least 90 days. While MIC90 of L80-T was > 4 mg/ml T, MIC90 of A80-T was 2 mg/ml T for all S. aureus strains. The antibiofilm formation activity was maximal for A80-T, while L80-T did not inhibit biofilm formation compared to untreated control. A80-T significantly decreased the biomass of preformed biofilms of S. aureus ATCC 25,923 strain and of 3 of the 4 clinical MRSA isolates at 4 mg/ml T. It was found that the viability of J774A.1 macrophages was decreased significantly upon 24 h incubation with A80-T, L80-T and T emulsion at 0.4 mg/ml T. These results show that from 0.4 mg/ml T, a value lower than MIC90 and the one displaying antibiofilm activity, with independence of its formulation, T significantly decreased the macrophages viability. CONCLUSION: Overall, because of its lower MIC90 against planktonic bacteria, higher antibiofilm formation capacity and stability during storage, A80-T resulted better antibacterial agent than T emulsion and L80-T. These results open new avenues to explode the A80-T antimicrobial intracellular activity.

Novel imiquimod nanovesicles for topical vaccination.

Development of needle and pain free noninvasive immunization procedures is a top priority for public health agencies. In this work the topical adjuvant activity of the immunomodulator imiquimod (IMQ) carried by ultradeformable archaeosomes (UDA2) (nanovesicles containing sn-2,3 ether linked phytanyl saturated archaeolipids) was surveyed and compared with that of ultradeformable liposomes lacking archaeolipids (UDL2) and free IMQ, using the model antigen ovalbumin and a seasonal influenza vaccine in Balb/c mice. UDA2 (250 ± 94 nm, -26 ± 4 mV Z potential) induced higher IMQ accumulation in human skin and higher production of TNF-α and IL-6 by macrophages and keratinocytes than free IMQ and UDL2. Mixed with ovalbumin, UDA2 was more efficient at generating cellular response, as measured by an increase in serum IgG2a and INF-γ production by splenocytes, compared with free IMQ and UDL2. Moreover, mixed with a seasonal influenza vaccine UDA2 produced same IgG titers and IgG2a/IgG1 isotypes ratio (≈1) than the subcutaneously administered influenza vaccine. Topical UDA2 however, induced highest stimulation index and INF-γ levels by splenocytes. UDA2 might be a promising adjuvant for topical immunization, since it produced cell-biased systemic response with ≈ 13-fold lower IMQ dose than the delivered as the commercial IMQ cream, Aldara.

Effect of circadian variation on neutrophil mobilization to the peripheral blood in benign constitutional neutropenia.

Benign constitutional neutropenia (BCN) is an asymptomatic condition characterized by mild chronic neutropenia in patients with no history of recurrent infections. Most patients are referred for further testing, increasing health care costs. We present an alternative form of assessment of individuals with BCN based on neutrophil circadian variation. The objective of this study was to evaluate whether circadian variations of neutrophil counts would result in neutrophil values higher than neutropenia threshold in individuals with BCN. Absolute neutrophil counts (ANCs) were evaluated in 102 patients with BCN (<1500 cells/µL) and 60 age- and sex-matched controls. Paired blood counts were performed in the early morning and in the early afternoon. We observed a significant difference between morning and afternoon ANC in BCN patients (879 cells/µL, 95% CI 745-1028, p < 0.001), as well as in the controls (619 cells/µL, 95% CI 443-796, p < 0.001). The ANC increase between the two evaluations was significantly greater in BCN patients compared with controls: 83% and 27%, respectively (p < 0.001). The ANC increment was higher in the groups with a lower morning ANC: 105%, 63%, and 27% in the <1000, 1000-1500, and >1500 cells/µL groups, respectively (p < 0.001). Of all BCN patients, 73% presented with >1500 cells/µL in the afternoon and 16% improved from the <1000 cells/µL to the 1000-1500 cells/µL category. In conclusion, neutrophil circadian variation measurement allows many BCN patients to be excluded from the neutropenia threshold. We identified a simple, easy, and feasible way to assess neutrophil reserve in patients with BCN with a potential reduction in costs of the assessment.