Identification of host cell factors required for intoxication through use of modified cholera toxin

We describe a novel labeling strategy to site-specifically attach fluorophores, biotin, and proteins to the C terminus of the A1 subunit (CTA1) of cholera toxin (CTx) in an otherwise correctly assembled and active CTx complex. Using a biotinylated N-linked glycosylation reporter peptide attached to CTA1, we provide direct evidence that ∼12% of the internalized CTA1 pool reaches the ER. We also explored the sortase labeling method to attach the catalytic subunit of diphtheria toxin as a toxic warhead to CTA1, thus converting CTx into a cytolethal toxin. This new toxin conjugate enabled us to conduct a genetic screen in human cells, which identified ST3GAL5, SLC35A2, B3GALT4, UGCG, and ELF4 as genes essential for CTx intoxication. The first four encode proteins involved in the synthesis of gangliosides, which are known receptors for CTx. Identification and isolation of the ST3GAL5 and SLC35A2 mutant clonal cells uncover a previously unappreciated differential contribution of gangliosides to intoxication by CTx.Fundação para a Ciência e a Tecnologia (Fellowship

Suppressing effect of CMPPE, a new positive allosteric modulator of the GABAB receptor, on alcohol self-administration and reinstatement of alcohol seeking in rats

Positive allosteric modulators (PAMs) of the GABAB receptor constitute a class of pharmacological agents gaining increasing attention in the alcohol research field because of their ability to suppress several alcohol-related behaviors in rodents. CMPPE is a novel GABAB PAM, still limitedly characterized in vivo. It was therefore of interest to test its ability to affect operant, oral self-administration of alcohol and cueinduced reinstatement of alcohol seeking in alcohol-preferring rats. To this end, female Sardinian alcohol-preferring (sP) rats were trained to lever-respond for alcohol (15% v/v) under the fixed ratio (FR) 5 (FR5) schedule of reinforcement. Once lever-responding had stabilized, rats were exposed to test sessions (under the FR5 [Experiment 1] and progressive ratio [PR; Experiment 2] schedules of reinforcement) preceded by treatment with CMPPE (0, 2.5, 5, and 10 mg/kg; intraperitoneally [i.p.]). In Experiment 3, once lever-responding had stabilized, rats underwent an extinction responding phase and then a single reinstatement session during which lever-responding was resumed by the non-contingent presentation of a complex of alcohol-associated cues; CMPPE (0, 2.5, 5, and 10 mg/kg; i.p.) was administered before the reinstatement session. Selectivity of CMPPE actionwas assessed by evaluating the effect of CMPPE (0, 2.5, 5, and 10 mg/kg; i.p.) on self-administration of a chocolate solution in male Wistar rats (Experiment 4). In Experiments 1 and 2, treatment with 5 and 10 mg/kg CMPPE reduced lever-responding and breakpoint for alcohol. In Experiment 3, treatment with 5 and 10 mg/kg CMPPE suppressed reinstatement of alcohol seeking. In Experiment 4, no dose of CMPPE affected lever-responding for the chocolate solution. These results extend to CMPPE the ability of all previously tested GABAB PAMs to affect alcohol-motivated behaviors in rodents and confirm that these effects are a shared feature of the entire class of GABAB PAMs. This conclusion is of relevance in view of the forthcoming transition of GABAB PAMs to clinical testing

Octopamine modulates the activity of motoneurons related to calling behavior in the gypsy moth Lymantria dispar

A morphofunctional investigation of the different neuronal subpopulations projecting through each of the nerves IV–VI emerging bilaterally from the terminal abdominal ganglion (TAG) was correlated with the octopaminergic activity in the ganglion that controls the ovipositor movements associated with calling behavior in the female gypsy moth Lymantria dispar. Tetramethylrodamine‐dextran backfills from nerve stumps resulted in a relatively low number of TAG projections, ranging from 12 to 13 for nerve pair IV, 12 to 14 for nerve pair V, and 8 to 9 for nerve pair VI. Furthermore, as assessed by electrophysiological recordings, a number of fibers within each of these nerves displays spontaneous tonic activity, also when the ganglion is fully disconnected from the ventral nerve cord (VNC). Octopamine (OA) applications to the TAG strongly enhanced the activity of these nerves, either by increasing the firing rate of a number of spontaneously firing units or by recruiting new ones. This octopaminergic activity affected calling behavior, and specifically the muscle activity leading to cycling extensions of the intersegmental membrane (IM) between segments VIII and IX (ovipositor). Our results indicate that in the female gypsy moth the octopaminergic neural activity of the TAG is coupled with extensions and retractions of IM for the purpose of releasing pheromone, where motor units innervated by nerve pair IV appear antagonistic with respect to those innervated by nerve pair V

The role of Labrasol® in the enhancement of the cutaneous bioavailability of minoxidil in phospholipid Vesicles

In this work we focused on how the concentration of Labrasol® and preparation method of phospholipid vesicles might influence their physico-chemical features and skin delivery performances. Penetration Enhancer-containing Vesicles (PEVs) were prepared using caprylcaproyl macrogol-8 glyceride (Labrasol®, HLB 14), a solubilizer/penetration enhancer for topical formulations. A cheap and unpurified commercial mixture of phospholipids, fatty acids and triglycerides (Phospholipon® 50) was used to incorporate minoxidil (2% w/v). During the preparation, labrasol was added at different concentrations in the lipid phase or mixed with the water phase. The film hydration method was used, followed by sonication, when appropriate. The effects that these variables might have on PEVs colloidal characteristics were investigated. Multi- and oligolamellar vesicles were obtained, depending on the production procedure, around 100 nm, negatively charged (~ -60 mV), able to incorporate minoxidil in good yields (up to 74%), and stable during storage. Further, vesicles were evaluated for ex vivo (trans)dermal delivery through new born pig skin, showing improved minoxidil local accumulation, influenced by labrasol concentration and insertion in the aqueous or lipid phase of the vesicular formulations. Overall, it can be assumed that PEVs enhanced drug transport through the skin barrier thanks to a synergic effect of vesicles and labrasol. The proposed approach based on vesicular nanocarriers may hold promising therapeutic value for treating hair growth disorders, by increasing cutaneous minoxidil bioavailability and reducing transdermal permeation

Exposure to an enriched environment reduces alcohol self-administration in Sardinian alcohol-preferring rats

: Living in an enriched environment (EE) produces a notable impact on several rodent behaviors, including those motivated by drugs of abuse. This picture is somewhat less clear when referring to alcohol-motivated behaviors. With the intent of contributing to this research field with data from one of the few rat lines selectively bred for excessive alcohol consumption, the present study investigated the effect of EE on operant oral alcohol self-administration in Sardinian alcohol-preferring (sP) rats. Starting from Postnatal Day (PND) 21, male sP rats were kept under 3 different housing conditions: impoverished environment (IE; single housing in shoebox-like cages with no environmental enrichment); standard environment (SE; small colony cages with 3 rats and no environmental enrichment); EE (large colony cages with 6 rats and multiple elements of environmental enrichment, including 2 floors, ladders, maze, running wheels, and shelter). From PND 60, rats were exposed to different phases of shaping and training of alcohol self-administration. IE, SE, and EE rats were then compared under (i) fixed ratio (FR) 4 (FR4) schedule of alcohol reinforcement for 20 daily sessions and (ii) progressive ratio (PR) schedule of alcohol reinforcement in a final single session. Acquisition of the lever-responding task (shaping) was slower in EE than IE and SE rats, as the likely consequence of a "devaluation" of the novel stimuli provided by the operant chamber in comparison to those to which EE rats were continuously exposed in their homecage or an alteration, induced by EE, of the rat "emotionality" state when facing the novel environment represented by the operant chamber. Training of alcohol self-administration was slower in EE than IE rats, with SE rats displaying intermediate values. A similar ranking order (IE>SE>EE) was also observed in number of lever-responses for alcohol, amount of self-administered alcohol, and breakpoint for alcohol under FR4 and PR schedules of reinforcement. These data suggest that living in a complex environment reduced the reinforcing and motivational properties of alcohol in sP rats. These results are interpreted in terms of the reinforcing and motivational properties of the main components of EE (i.e., social interactions, physical activities, exploration, novelty) substituting, at least partially, for those of alcohol

Abdominal Lymphadenopathy: Hypothesize Cat-Scratch Disease and Avoid Abdominal Excisional Biopsy

The finding of lymphadenopathy is usually the consequence of a benign infection, although a neoplastic origin must always be excluded. Through a careful anamnesis, physical examination, and serological tests several differential diagnoses are frequently possible. Nevertheless, sometimes an excisional biopsy of superficial lymph nodes is required, which is the best means to reach a definitive diagnosis. More concerns arise when lymphadenopathy is only abdominal/retroperitoneal: percutaneous biopsy is often inconclusive and the excisional node biopsy becomes a surgical procedure, certainly indicated in case of malignancy but avoidable in case of inflammatory diseases. We present the case of a 30-year-old man with a deep iliac lymphadenopathy who was evaluated at the Hematological Unit of Sapienza University of Rome. The enlargement of an iliac lymph node is quite unusual for an infectious disease. Although symptoms such as pain, fever, and chills suggested it was the case, cat-scratch disease was not hypothesized. Radiological investigations did not exclude a malignant disease and a laparoscopic excisional biopsy was scheduled, but the slight improvement of his spontaneous symptoms suggested a careful follow-up. Given the lack of disappearance of lymphadenopathy, the lack of diagnosis, and an ipsilateral superficial (inguinal) lymph node with similar ultrasonographic and radiological features, the patient underwent biopsy, which disclosed a diagnosis of cat-scratch disease, avoiding more invasive surgical procedures

Abdominal Lymphadenopathy: Hypothesize Cat-Scratch Disease and Avoid Abdominal Excisional Biopsy

The finding of lymphadenopathy is usually the consequence of a benign infection, although a neoplastic origin must always be excluded. Through a careful anamnesis, physical examination, and serological tests several differential diagnoses are frequently possible. Nevertheless, sometimes an excisional biopsy of superficial lymph nodes is required, which is the best means to reach a definitive diagnosis. More concerns arise when lymphadenopathy is only abdominal/retroperitoneal: percutaneous biopsy is often inconclusive and the excisional node biopsy becomes a surgical procedure, certainly indicated in case of malignancy but avoidable in case of inflammatory diseases. We present the case of a 30-year-old man with a deep iliac lymphadenopathy who was evaluated at the Hematological Unit of Sapienza University of Rome. The enlargement of an iliac lymph node is quite unusual for an infectious disease. Although symptoms such as pain, fever, and chills suggested it was the case, cat-scratch disease was not hypothesized. Radiological investigations did not exclude a malignant disease and a laparoscopic excisional biopsy was scheduled, but the slight improvement of his spontaneous symptoms suggested a careful follow-up. Given the lack of disappearance of lymphadenopathy, the lack of diagnosis, and an ipsilateral superficial (inguinal) lymph node with similar ultrasonographic and radiological features, the patient underwent biopsy, which disclosed a diagnosis of cat-scratch disease, avoiding more invasive surgical procedures

Physico-chemical characterization of succinyl chitosan-stabilized liposomes for the oral co-delivery of quercetin and resveratrol

In the present work, quercetin and resveratrol, natural polyphenols with strong antioxidant and anti-inflammatory properties, were co-loaded in polymer-associated liposomes conceived for oral delivery, by exploiting the potential of pH-sensitive succinyl-chitosan. Chitosan was succinylated, characterized by Nuclear Magnetic Resonance spectroscopy and Gel Permeation Chromatography, and used to form a protective shell on the surface of liposomes. The physico-chemical properties of the succinyl-chitosan liposomes were assessed by light scattering, zeta potential, cryogenic transmission electron microscopy, and small angle X-ray scattering. Small, spherical, uni- and bilamellar vesicles were produced. The succinyl-chitosan shell increased not only the physical stability of the vesicular system, as demonstrated by accelerated stability tests, but also the release of the polyphenols to a greater extent at pH 7.0, mimicking the intestinal environment. The proposed approach based on polyphenol vesicular formulations may be of value in the treatment of pre-cancerous/cancerous intestinal conditions associated with inflammation and oxidative stress