Biophysical investigation of biomolecules in bio-membrane models

2010 - 2011Many drugs are available for the treatment of systemic or superficial mycoses, but only a limited number of them are effective antifungal drugs, devoid of toxic and undesirable side effects. Therefore there remains an urgent need for a new generation of antifungal agents. The present work concerns the synthesis, the antifungal activity and the biophysical characterization of a set of linear and cyclic peptides (AMT1, cyclo-AMT1, AMT2, cyclo-AMT2, AMT3, cyclo-AMT3) including aminoacids characteristic of membrane-active antimicrobial peptides (AMP). The peptides were tested against different yeast species, and displayed general antifungal activity, with a therapeutically promising antifungal specificity against Cryptococcus neoformans. To shed light on the role played by the membrane cell in the antifungal activity an extensive biophysical study was carried out using different spectroscopic techniques. Our structural investigation provides data to exclude the ability of the peptides to penetrate the membrane of the fungal cell, highlighting their attitude to interact with the external surface of the bilayer. Taken together our data support the hypothesis that the membrane cell of the fungi may be an important platform for specific interactions of the synthesized peptides with more specific targets involved in the cell wall synthesis. Viral fusion glycoproteins present a membrane-proximal external region (MPER) which is usually rich in aromatic residues and presents a marked tendency to stably reside at the membrane interfaces, leading, through unknown mechanisms, to a destabilization of the bilayer structure. This step has been proposed to be fundamental for the fusion process between target membrane and viral envelope. In present work, we investigate the interaction between an octapeptide (C8) deriving from the MPER domain of gp36 of Feline Immunodeficiency Virus and different membrane models by combining experimental results obtained by Nuclear Magnetic Resonance, Electron Spin Resonance, Circular Dichroism and Fluorescence Spectroscopy with Molecular Dynamics simulations. Our data indicate that C8 binds to the lipid bilayer adsorbing onto the membrane surface without deep penetration. As a consequence of this interaction, the bilayer thickness decreases. The association of the peptide with the lipid membrane is driven by hydrogen bonds as well as hydrophobic interactions that the Trp side chains form with the lipid headgroups. Notably these interactions may be the key to interpret at molecular level the function played by Trp residues in all the fragments of viral envelope involved in fusion mechanism with target membrane. [edited by author]X n.s

A new record and biological evidence supporting the establishment of Beryx splendens (Actinopterygii: Beryciformes: berycidae) in the western Mediterranean basin

A new specimen of splendid alfonsino, Beryx splendens Lowe, 1834, was caught by trawling in July 2016 in the Ligurian Sea at the depth of 350 m, in the proximity of a submarine canyon. It represents the 10th documented record of B. splendens in the Mediterranean. Because of the rarity of the records in the basin, there could be doubts whether to consider or not such species as established in the area. However, some fndings may support the hypothesis of the possible establishment of the species in the Mediterranean Sea. For example, the stomach of the specimen was nearly full, with rests of one crustacean decapod, one fsh, and some cephalopods, which are commonly reported prey items for the species. Macroscopic observation and histological analysis of the gonads revealed that the specimen was a male in an advanced stage of gonadal development. Finally, the coherence of habitat type and prey items with that of extra-Mediterranean populations coupled with gonadal maturation consistent with the observations on other specimens caught in the Mediterranean

883 An anti-carcinoma monoclonal antibody (mAb) NEO-201 can also target human acute myeloid leukemia (AML) cell lines in vitro

BackgroundNEO-201 is an IgG1 mAb targeting variants of CEACAM5/6 and has demonstrated tumor sensitivity and specificity in epithelial cells. Functional analysis has revealed that NEO-201 can engage innate immune effector mechanisms including ADCC and CDC to directly kill tumor cells expressing its target. A recent Phase 1 clinical trial at the NCI has determined both safety and recommended Phase 2 dosing. We have also seen the expression of the NEO-201 target on hematologic cells, specifically Tregs and neutrophils. Due to epitope being expressed both on malignant epithelial cells as well as several hematologic cells, we designed this study to explore the reactivity of NEO-201 against hematological neoplastic cells in vitro.MethodsPhenotypic analysis was conducted by flow cytometry. Cell lines used were six AML (HL60, U937, MOLM13, AML2, IMS-M2 and OCL-AML3), two multiple myelomas (MM) (OPM2, MM1.S), two acute lymphoblastic leukemia (ALL) (SUP-B15, RPMI8402) and four mantle cell lymphoma (MCL) (Jeko-1, Z138, JVM2 and JVM13). Markers used for flow cytometry analysis were CD15, CD45, CD38, CD138, CD14, CD19 and NEO-201. Functional analysis was performed by evaluating the ability of NEO-201 to mediate ADCC activity against AML cell lines using human NK cells as effector cells.Results5 of 6 AML cell lines tested bind to NEO-201 and the% of positive cells were 47%, 99.5%,100%,100% and 97.8% for HL60, U937, MOLM13, AML3 and IMS-M2, respectively. The% of positive cells in the two MM cell line were 99% and 18% for OPM2 and MM1.S, respectively. NEO-201 binding was not detected in the two ALL and the four MCL cell lines tested. Functional analysis has demonstrated that NEO-201 can mediate ADCC activity against the AML cell line (HL60) tested.ConclusionsThis study demonstrates that NEO-201 mAb's target is expressed in most of the AML cell lines tested in vitro. In addition, we have shown it can mediate ADCC activity against HL60 cells (AML). Together, these findings provide a rationale for further investigation of the role of NEO-201 in AML as well as MM, further exploring patient PBMCs and bone marrow samples

The Arrangement of the Peripheral Olfactory System of Pleuragramma antarcticum: A Well-Exploited Small Sensor, an Aided Water Flow, and a Prominent Effort in Primary Signal Elaboration

The olfactory system is constituted in a consistent way across vertebrates. Nasal structures allow water/air to enter an olfactory cavity, conveying the odorants to a sensory surface. There, the olfactory neurons form, with their axons, a sensory nerve projecting to the telencephalic zone\u2014named the olfactory bulb. This organization comes with many different arrangements, whose meaning is still a matter of debate. A morphological description of the olfactory system of many teleost species is present in the literature; nevertheless, morphological investigations rarely provide a quantitative approach that would help to provide a deeper understanding of the structures where sensory and elaborating events happen. In this study, the peripheral olfactory system of the Antarctic silverfish, which is a keystone species in coastal Antarctica ecosystems, has also been described, employing some quantitative methods. The olfactory chamber of this species is connected to accessory nasal sacs, which probably aid water movements in the chamber; thus, the head of the Antarctic silverfish is specialized to assure that the olfactory organ keeps in contact with a large volume of water\u2014even when the fish is not actively swimming. Each olfactory organ, shaped like an asymmetric rosette, has, in adult fish, a sensory surface area of about 25 mm2, while each olfactory bulb contains about 100,000 neurons. The sensory surface area and the number of neurons in the primary olfactory brain region show that this fish invests energy in the detection and elaboration of olfactory signals and allow comparisons among different species. The mouse, for example\u2014which is considered a macrosmatic vertebrate\u2014has a sensory surface area of the same order of magnitude as that of the Antarctic silverfish, but ten times more neurons in the olfactory bulb. Catsharks, on the other hand, have a sensory surface area that is two orders of magnitude higher than that of the Antarctic silverfish, while the number of neurons has the same order of magnitude. The Antarctic silverfish is therefore likely to rely considerably on olfaction

Beta-amyloid-acetylcholine structural interaction: evidence for neuroprotective effects of acetylcholine in neural cells

Alzheimer’s disease (AD) is regarded as a multifactorial disease characterized by a complex pathogenesis including a cholinergic deficit - due to degeneration of cholinergic projections from the basal forebrain - and the extracellular accumulation of amyloid beta (Aβ) peptide. Aβ containing 39 to 42 amino acids is the predominant component of the senile plaques that, together with neurofibrillary tangles, are regarded as the neuropathological hallmarks of AD (Sorrentino et al. 2014). Aβ may assume different conformations changing from random coil or α-helical monomers to β-sheet structures forming toxic oligomers and/or β-sheet mature fibrils. In this framework, we studied the effect of acetylcholine (ACh) on the conformation of Aβ by circular dichroism analysis. Moreover we investigated the ability of ACh to protect neuronal cells from the toxic action of amyloid peptide and to modulate the neuroinflammatory response occurring via the phospholipase A2 (PLA2). Results show that the amount of Aβ(25-35) β-strand raised linearly in absence of ACh, whereas it remained almost constant in presence of ACh. In addition, in a micelle solution mimicking the membrane environment ACh was found effective in increasing and stabilizing the soluble and not toxic helical content of Aβ(25-35) suggesting that ACh is capable to preserve the soluble form of Aβ(25-35), reducing the incipit of Aβ aggregation. In order to assess the neuro-protective ability of ACh against toxic Aβ(25-35) accumulation, we used neural cell (NCC) cultures containing both astrocytes and glial cells prepared from brains embryos from timed pregnant Wistar rats and infused ACh for 48h. By immunostaining, we observed that ACh reduced Aβ(25-35)-induced cell death. Then, we tested the protective effect of ACh on inflammation induced by Aβ administration. NCC were challenged with Aβ(25-35) in the presence and absence of ACh and immunostained for astroglial and neuronal markers: results showed a reduction of the morphological features of astrogliosys in ACh treated cells. PLA2 expression analysis corroborated these data also underlying that ACh can negatively regulate inflammation pathways in glial cells

Mantle Cell Lymphoma of Mucosa-Associated Lymphoid Tissue:A European Mantle Cell Lymphoma Network Study

While classical nodal mantle cell lymphoma (cMCL) is often associated with involvement of multiple extranodal sites, isolated extranodal disease (ED) at the time of diagnosis is a rare event; data on the outcome of these forms are lacking. On behalf of the European MCL Network, we conducted a retrospective analysis on the clinical characteristics and outcomes of MCL presenting with isolated or predominant ED (MALT MCL). We collected data on 127 patients with MALT MCL diagnosed from 1998 to 2015: 78 patients (61%) were male with a median age of 65 years. The involved sites include: upper airways + Waldeyer ring (40; 32%), gastrointestinal tract (32; 25%), ocular adnexa (17; 13%), oral cavity and salivary glands (17; 13%) and others (13; 1%); 7 patients showed multiple extranodal sites. The median follow-up was 80 months (range: 6–182), 5-year progression-free survival (PFS) was 45% (95% CI: 35–54) and 5-year overall survival (OS) was 71% (95% CI: 62–79). In an explorative setting, we compared MALT MCL with a group of 128 cMCL patients: MALT MCL patients showed a significantly longer PFS and OS compared with nodal cMCL; with a median PFS of 4.5 years vs 2.8 years (p = 0.001) and median OS of 9.8 years vs 6.9 years (p = 0.018), respectively. Patients with MALT MCL at diagnosis showed a more favorable prognosis and indolent course than classical nodal type. This clinical variant of MCL should be acknowledged to avoid possible over-treatment

SARS-CoV-2 multi-variant rapid detector based on graphene transistor functionalized with an engineered dimeric ACE2 receptor

Reliable point-of-care (POC) rapid tests are crucial to detect infection and contain the spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The emergence of several variants of concern (VOC) can reduce binding affinity to diagnostic antibodies, limiting the efficacy of the currently adopted tests, while showing unaltered or increased affinity for the host receptor, angiotensin converting enzyme 2 (ACE2). We present a graphene field-effect transistor (gFET) biosensor design, which exploits the Spike-ACE2 interaction, the crucial step for SARS-CoV-2 infection. Extensive computational analyses show that a chimeric ACE2-Fragment crystallizable (ACE2-Fc) construct mimics the native receptor dimeric conformation. ACE2-Fc functionalized gFET allows in vitro detection of the trimeric Spike protein, outperforming functionalization with a diagnostic antibody or with the soluble ACE2 portion, resulting in a sensitivity of 20 pg/mL. Our miniaturized POC biosensor successfully detects B.1.610 (pre-VOC), Alpha, Beta, Gamma, Delta, Omicron (i.e., BA.1, BA.2, BA.4, BA.5, BA.2.75 and BQ.1) variants in isolated viruses and patient's clinical nasopharyngeal swabs. The biosensor reached a Limit Of Detection (LOD) of 65 cps/mL in swab specimens of Omicron BA.5. Our approach paves the way for a new and reusable class of highly sensitive, rapid and variant-robust SARS-CoV-2 detection systems