Preliminary dosimetric analysis of DOTA-folate radiopharmaceutical radiolabelled with 47Sc produced through natV(p,x)47Sc cyclotron irradiation.

47Sc is one of the most promising theranostic radionuclides, thanks to its low energy γ-ray emission (159 keV), suitable for single photon emission computed tomography imaging and its intense ß - emission, useful for tumour treatment. Despite promising preclinical results, the translation of 47Sc-therapeutic agents to the clinic is hampered by its limited availability. Among different 47Sc-production routes currently being investigated, the natV(p,x)47Sc reaction has proved to be of particular interest, thanks to the low-cost and easy availability on the market of natV material and the diffusion of medium energy proton cyclotrons. However, the cross section of this specific nuclear reaction is quite low and small amounts of Sc-contaminants are co-produced at energies E P ≤ 45 MeV, namely 48Sc and 46Sc. The main concern with these Sc-contaminants is their contribution to the patient absorbed dose. For such a reason, the absorbed dose contributions to healthy organs and the effective dose contributions by the three radioisotopes, 48Sc, 47Sc and 46Sc, were evaluated using DOTA-folate conjugate (cm10) as an example of radiopharmaceutical product. Considering as acceptable the limits of 99% for the radionuclidic purity and 10% for the contribution of radioactive Sc-contaminants to the total effective dose after 47Sc-cm10 injection, it was obtained that proton beam energies below 35 MeV must be used to produce 47Sc through irradiation of a natV target.

Two-pore and TRPML cation channels: Regulators of phagocytosis, autophagy and lysosomal exocytosis.

The old Greek saying "Panta Rhei" ("everything flows") is true for all life and all living things in general. It also becomes nicely evident when looking closely into cells. There, material from the extracellular space is taken up by endocytic processes and transported to endosomes where it is sorted either for recycling or degradation. Cargo is also packaged for export through exocytosis involving the Golgi network, lysosomes and other organelles. Everything in this system is in constant motion and many proteins are necessary to coordinate transport along the different intracellular pathways to avoid chaos. Among these proteins are ion channels., in particular TRPML channels (mucolipins) and two-pore channels (TPCs) which reside on endosomal and lysosomal membranes to speed up movement between organelles, e.g. by regulating fusion and fission; they help readjust pH and osmolarity changes due to such processes, or they promote exocytosis of export material. Pathophysiologically, these channels are involved in neurodegenerative, metabolic, retinal and infectious diseases, cancer, pigmentation defects, and immune cell function, and thus have been proposed as novel pharmacological targets, e.g. for the treatment of lysosomal storage disorders, Duchenne muscular dystrophy, or different types of cancer. Here, we discuss the similarities but also differences of TPCs and TRPMLs in regulating phagocytosis, autophagy and lysosomal exocytosis, and we address the contradictions and open questions in the field relating to the roles TPCs and TRPMLs play in these different processes.

TRPML1 links lysosomal calcium to autophagosome biogenesis through the activation of the CaMKKß/VPS34 pathway.

The lysosomal calcium channel TRPML1, whose mutations cause the lysosomal storage disorder (LSD) mucolipidosis type IV (MLIV), contributes to upregulate autophagic genes by inducing the nuclear translocation of the transcription factor EB (TFEB). Here we show that TRPML1 activation also induces autophagic vesicle (AV) biogenesis through the generation of phosphatidylinositol 3-phosphate (PI3P) and the recruitment of essential PI3P-binding proteins to the nascent phagophore in a TFEB-independent manner. Thus, TRPML1 activation of phagophore formation requires the calcium-dependent kinase CaMKKß and AMPK, which increase the activation of ULK1 and VPS34 autophagic protein complexes. Consistently, cells from MLIV patients show a reduced recruitment of PI3P-binding proteins to the phagophore during autophagy induction, suggesting that altered AV biogenesis is part of the pathological features of this disease. Together, we show that TRPML1 is a multistep regulator of autophagy that may be targeted for therapeutic purposes to treat LSDs and other autophagic disorders.

Radioisotopic purity and imaging properties of cyclotron-produced 99mTc using direct 100Mo(p,2n) reaction.

Evaluation of the radioisotopic purity of technetium-99m (99mTc) produced in GBq amounts by proton bombardment of enriched molibdenum-100 (100Mo) metallic targets at low proton energies (i.e. within 15-20 MeV) is conducted. This energy range was chosen since it is easily achievable by many conventional medical cyclotrons already available in the nuclear medicine departments of hospitals. The main motivation for such a study is in the framework of the research activities at the international level that have been conducted over the last few years to develop alternative production routes for the most widespread radioisotope used in medical imaging. The analysis of technetium isotopes and isomeric states (9xTc) present in the pertechnetate saline Na99mTcO4 solutions, obtained after the extraction/purification procedure, reveals radionuclidic purity levels basically in compliance with the limits recently issued by European Pharmacopoeia 9.3 (2018 Sodium pertechnetate (99mTc) injection 4801-3). Moreover, the impact of 9xTc contaminant nuclides on the final image quality is thoroughly evaluated, analyzing the emitted high-energy gamma rays and their influence on the image quality. The spatial resolution of images from cyclotron-produced 99mTc acquired with a mini-gamma camera was determined and compared with that obtained using technetium-99m solutions eluted from standard 99Mo/99mTc generators. The effect of the increased image background contribution due to Compton-scattered higher-energy gamma rays (E γ > 200 keV), which could cause image-contrast deterioration, was also studied. It is concluded that, due to the high radionuclidic purity of cyclotron-produced 99mTc using 100Mo(p,2n)99mTc reaction at a proton beam energy in the range 15.7-19.4 MeV, the resulting image properties are well comparable with those from the generator-eluted 99mTc.

Hydrogels for biomedical applications from glycol chitosan and PEG diglycidyl ether exhibit pro-angiogenic and antibacterial activity.

We aimed at producing a hydrogel from a chitosan (CS) derivative soluble in physiological conditions to avoid any purification step thus allowing to use the materials also as an in-situ forming material. So, we crosslinked glycol chitosan (GCS) with poly(ethylene glycol) diglycidyl ether (PEGDE) in water at 37 °C. The scaffolds, referred as GCS-PEG, were specifically designed to be used as wound dressing materials as such (after crosslinking) or as in-situ forming materials. Different amounts of PEGDE were tested. The obtained scaffolds showed macroscopic pores and a tailorable swelling in water by controlling the crosslinking degree. Moreover, GCS-PEG scaffolds displayed a significant antimicrobial activity against Staphylococcus aureus. In-vivo study using the chick embryo choriallantoic membrane resulted in a highly pronounced pro-angiogenic activity suggesting important tissue regeneration properties. Moreover, the employed materials are commercially available, no organic solvents are required and the scaling up is quite predictable.

Rocuronio-sugammadex para el manejo de la terapia electroconvulsiva en el síndrome neuroléptico maligno, a propósito de un caso / Rocuronium-sugammadex for electroconvulsive therapy management in neuroleptic malignant syndrome: a case report

Los neurolépticos son un grupo de medicamentos ampliamente empleados en el tratamiento de cuadros psicóticos, entre sus efectos adversos cabe destacar la posibilidad de desencadenar un síndrome neuroléptico maligno (SNM). El diagnóstico del SNM se determina por exclusión y su manejo terapéutico inicial será la retirada de los neurolépticos junto a la administración de benzodiacepinas y terapia electroconvulsiva (TEC). La TEC representa una efectiva opción terapéutica en estos pacientes así como en aquellos casos que se obtenga una respuesta escasa al manejo con medicamentos antipsicóticos. Revisamos las alternativas terapéuticas y las implicaciones anestésicas que conlleva manejar un paciente programado para TEC, diagnosticado de esquizofrenia paranoide, en el contexto de SNM (AU)

Neuroleptics are a group of drugs widely used in the treatment of psychotic symptoms. Among their adverse effects is the ability to trigger a neuroleptic malignant syndrome (NMS). The diagnosis of NMS is determined by exclusion, and its initial therapeutic management should be the withdrawal of neuroleptics, the administration of benzodiazepines, and electroconvulsive therapy (ECT). ECT is an effective treatment in these patients, and in those cases with a poor response to treatment with antipsychotic drugs. A review is presented on the treatment options and anaesthetic implications of ECT used to handle a patient diagnosed with paranoid schizophrenia in the context of NMS (AU)

Energy decay in a tapped granular column: Can a one-dimensional toy model provide insight into fully three-dimensional systems?

The decay of energy within particulate media subjected to an impulse is an issue of significant scientific interest, but also one with numerous important practical applications. In this paper, we study the dynamics of a granular system exposed to energetic impulses in the form of discrete taps from a solid surface. By considering a one-dimensional toy system, we develop a simple theory, which successfully describes the energy decay within the system following exposure to an impulse. We then extend this theory so as to make it applicable also to more realistic, three-dimensional granular systems, assessing the validity of the model through direct comparison with discrete particle method simulations. The theoretical form presented possesses several notable consequences; in particular, it is demonstrated that for suitably large systems, effects due to the bounding walls may be entirely neglected. We also establish the existence of a threshold system size above which a granular bed may be considered fully three dimensional.

Rocuronium-sugammadex for electroconvulsive therapy management in neuroleptic malignant syndrome: A case report. / Rocuronio-sugammadex para el manejo de la terapia electroconvulsiva en el síndrome neuroléptico maligno, a propósito de un caso.

Neuroleptics are a group of drugs widely used in the treatment of psychotic symptoms. Among their adverse effects is the ability to trigger a neuroleptic malignant syndrome (NMS). The diagnosis of NMS is determined by exclusion, and its initial therapeutic management should be the withdrawal of neuroleptics, the administration of benzodiazepines, and electroconvulsive therapy (ECT). ECT is an effective treatment in these patients, and in those cases with a poor response to treatment with antipsychotic drugs. A review is presented on the treatment options and anaesthetic implications of ECT used to handle a patient diagnosed with paranoid schizophrenia in the context of NMS.

The DisVis and PowerFit Web Servers: Explorative and Integrative Modeling of Biomolecular Complexes.

Structure determination of complex molecular machines requires a combination of an increasing number of experimental methods with highly specialized software geared toward each data source to properly handle the gathered data. Recently, we introduced the two software packages PowerFit and DisVis. These combine high-resolution structures of atomic subunits with density maps from cryo-electron microscopy or distance restraints, typically acquired by chemical cross-linking coupled with mass spectrometry, respectively. Here, we report on recent advances in both GPGPU-accelerated software packages: PowerFit is a tool for rigid body fitting of atomic structures in cryo-electron density maps and has been updated to also output reliability indicators for the success of fitting, through the use of the Fisher z-transformation and associated confidence intervals; DisVis aims at quantifying the information content of distance restraints and identifying false-positive restraints. We extended its analysis capabilities to include an analysis of putative interface residues and to output an average shape representing the putative location of the ligand. To facilitate their use by a broad community, they have been implemented as web portals harvesting both local CPU resources and GPGPU-accelerated EGI grid resources. They offer user-friendly interfaces, while minimizing computational requirements, and provide a first interactive view of the results. The portals can be accessed freely after registration via http://milou.science.uu.nl/services/DISVIS and http://milou.science.uu.nl/services/POWERFIT.

Modeling Meropenem Treatment, Alone and in Combination with Daptomycin, for KPC-Producing Klebsiella pneumoniae Strains with Unusually Low Carbapenem MICs.

Klebsiella pneumoniae strains producing K. pneumoniae carbapenemase (KPC) cause serious infections in debilitated and immunocompromised patients and are associated with prolonged hospital stays and increased mortality rates. Daptomycin is a lipopeptide used against Staphylococcus aureus infection and considered inactive against Gram-negative bacteria. We investigated the effectiveness of a daptomycin-meropenem combination by synergy kill curve and a pharmacokinetic/pharmacodynamic model. The combination may represent a novel therapeutic strategy against infections caused by KPC-producing K. pneumoniae strains.

Enhanced solubility and antibacterial activity of lipophilic fluoro-substituted N-benzoyl-2-aminobenzothiazoles by complexation with ß-cyclodextrins.

Some lipophilic fluoro-substituted N-benzoyl-2-aminobenzothiazole antibacterial agents have been evaluated for their activity in the presence of cyclodextrins (CDs) containing aqueous solutions where CDs are adopted as solubilizing excipients for improving the poor water solubility of these compounds. For such purpose both the natural ß-CD and one of FDA/EMA approved CDs for parenteral use (i.e. HP-ß-CD) have been employed. The solubility rank order observed was accounted for by thermal analysis (Differential Scanning Calorimetry) and FT-IR spectroscopy. The most promising compound was subjected to further NMR spectroscopic studies and molecular modelling simulations to verify the interactions between the guest molecule and the CD cavity. The assessment of the antibacterial activity of such compounds against selected Gram positive and Gram negative bacterial strains clearly showed that their antimicrobial effectiveness may, quite in all instances, be positively affected by complexation with ß-CD and HP-ß-CD. These results, which are in some ways in contrast with those already reported in the literature, are herein discussed on the basis of plausible mechanisms. Moreover, this investigation also reveals that the described methodology of complexing both lipophilic and hydrophilic antimicrobial agents with CDs may be an useful approach to enhance their effectiveness as well as a promising strategy to overcome even the microbial resistance problem.

Improvement of diaphragmatic performance through orthotopic application of decellularized extracellular matrix patch.

Muscle tissue engineering can provide support to large congenital skeletal muscle defects using scaffolds able to allow cell migration, proliferation and differentiation. Acellular extracellular matrix (ECM) scaffold can generate a positive inflammatory response through the activation of anti-inflammatory T-cell populations and M2 polarized macrophages that together lead to a local pro-regenerative environment. This immunoregulatory effect is maintained when acellular matrices are transplanted in a xenogeneic setting, but it remains unclear whether it can be therapeutic in a model of muscle diseases. We demonstrated here for the first time that orthotopic transplantation of a decellularized diaphragmatic muscle from wild animals promoted tissue functional recovery in an established atrophic mouse model. In particular, ECM supported a local immunoresponse activating a pro-regenerative environment and stimulating host muscle progenitor cell activation and migration. These results indicate that acellular scaffolds may represent a suitable regenerative medicine option for improving performance of diseased muscles.

Maximizing energy transfer in vibrofluidized granular systems.

Using discrete particle simulations validated by experimental data acquired using the positron emission particle tracking technique, we study the efficiency of energy transfer from a vibrating wall to a system of discrete, macroscopic particles. We demonstrate that even for a fixed input energy from the wall, energy conveyed to the granular system under excitation may vary significantly dependent on the frequency and amplitude of the driving oscillations. We investigate the manner in which the efficiency with which energy is transferred to the system depends on the system variables and determine the key control parameters governing the optimization of this energy transfer. A mechanism capable of explaining our results is proposed, and the implications of our findings in the research field of granular dynamics as well as their possible utilization in industrial applications are discussed.

Iron- and hemin-dependent gene expression of Porphyromonas gingivalis.

Although iron under anaerobic conditions is more accessible and highly reactive because of its reduced form, iron-dependent regulation is not well known in anaerobic bacteria. Here, we investigated iron- and hemin-dependent gene regulation in Porphyromonas gingivalis, an established periodontopathogen that primarily inhabits anaerobic pockets. Whole-genome microarrays of P. gingivalis genes were used to compare the levels of gene expression under iron-replete and iron-depleted conditions as well as under hemin-replete and hemin-depleted conditions. Under iron-depleted conditions, the expression of genes encoding proteins that participate in iron uptake and adhesion/invasion of host cells was increased, while that of genes encoding proteins involved in iron storage, energy metabolism, and electron transport was decreased. Interestingly, many of the genes with altered expression had no known function. Limiting the amount of hemin also resulted in a reduced expression of the genes encoding proteins involved in energy metabolism and electron transport. However, hemin also had a significant effect on many other biological processes such as oxidative stress protection and lipopolysaccharide synthesis. Overall, comparison of the data from iron-depleted conditions to those from hemin-depleted ones showed that although some regulation is through the iron derived from hemin, there also is significant distinct regulation through hemin only. Furthermore, our data showed that the molecular mechanisms of iron-dependent regulation are novel as the deletion of the putative Fur protein had no effect on the expression of iron-regulated genes. Finally, our functional studies demonstrated greater survivability of host cells in the presence of the iron-stressed bacterium than the iron-replete P. gingivalis cells. The major iron-regulated proteins encoded by PG1019-20 may play a role in this process as deletion of these sequences also resulted in reduced survival of the bacterium when grown with eukaryotic cells. Taken together, the results of this study demonstrated the utility of whole-genome microarray analysis for the identification of genes with altered expression profiles during varying growth conditions and provided a framework for the detailed analysis of the molecular mechanisms of iron and hemin acquisition, metabolism and virulence of P. gingivalis.

A single-chain fragment against prostate specific membrane antigen as a tool to build theranostic reagents for prostate cancer.

Prostate carcinoma is the most common non-cutaneous cancer in developed countries and represents the second leading cause of death. Early stage androgen dependent prostate carcinoma responds well to conventional therapies, but relatively few treatment options exist for patients with hormone-refractory prostate cancer. One of the most suitable targets for antibody-mediated approaches is prostate specific membrane antigen (PSMA) which is a well known tumour associated antigen. PSMA is a type II integral cell-surface membrane protein that is not secreted, and its expression density and enzymatic activity are increased progressively in prostate cancer compared to normal prostate epithelium, thereby making PSMA an ideal target for monoclonal antibody imaging and therapy. To obtain a small protein that can better penetrate tissue, we have engineered a single-chain variable fragment (scFv) starting from the variable heavy and light domains of the murine anti-PSMA monoclonal antibody D2B. scFvD2B was analysed in vitro for activity, stability, internalisation ability and in vivo for targeting specificity. Maintenance of function and immunoreactivity as well as extremely high radiolabelling efficiency and radiochemical purity were demonstrated by in vitro assays and under different experimental conditions. Despite its monovalent binding, scFvD2B retained a good strength of binding and was able to internalise around 40% of bound antigen. In vivo we showed its ability to specifically target only PSMA expressing prostate cancer xenografts. Due to these advantageous properties, scFvD2B has the potential to become a good theranostic reagent for early detection and therapy of prostate cancers.

Surgical strategy for the treatment of sporadic medullary thyroid carcinoma: our experience.

BACKGROUND: Medullary thyroid carcinoma (MTC) is a rare disease which accounts for approximately 5-9% of all thyroid cancers and originates from the calcitonin-screening parafollicular C cells. MTC can be divided into two subgroups: sporadic (75%) or inherited (25%). The majority of patients with invasive MTC have metastasis to regional lymph nodes at the time of diagnosis, as evidenced by the frequent finding of persistently elevated calcitonin levels after thyroidectomy and the high rates of recurrence in the cervical lymph nodes reported in retrospective studies. OBJECTIVES: The purpose of the study is to review our single institution's experience with MTC since 1998 and to evaluate surgical strategy, patterns of lymph node metastases and calcitonin response to compartment-oriented lymphadenectomy in patients with primary or recurrent sporadic medullary thyroid carcinoma. METHODS: A retrospective review of 26 patients treated for MTC at the "Antonio Cardarelli" Hospital referral center, in Naples, between 1998 and 2012. There were 18 female and 8 male patients, median age at presentation was 55 years, and median follow-up for survivors was 5 years. Total thyroidectomy was performed in all 26 patients; central compartment (CC) node dissection (level VI) in 12 (46%) patients; central plus lateral compartment (LC) node dissection (levels II, III, and IV) in 7 (27%) patients. 4 patients (15%) underwent reoperation for loco-regional recurrent/persistent MTC. Results. After a median post-surgical follow-up of 5 years (range 1-10 years), 63 % of patients were living disease-free, 15% were living with disease and/or persistently elevated calcitonin levels after surgery, 11% were deceased due to MTC and 11 % were lost to follow-up. CONCLUSIONS: We agree with most authors advocating for a total thyroidectomy and prophylactic central neck dissection in the setting of clinically detected MTC. Lateral neck dissection may be best reserved for patients with positive preoperative imaging. Nevertheless MTC has a high rate of lymph node metastases that are sub optimally detected preoperatively in the central compartment by neck ultrasound or intra-operatively by the surgeon, and reoperation is associated with a higher rate of surgical complications. In our limited experience, patients with thyroid confined nodular pathology, without nodal disease and unknown preoperative diagnosis of MTC, underwent only total thyroidectomy with a good prognosis.

Paclitaxel-hyaluronic acid for intravesical therapy of bacillus Calmette-Guérin refractory carcinoma in situ of the bladder: results of a phase I study.

PURPOSE: Carcinoma in situ represents high grade anaplasia of the bladder mucosa. Intravesical immunotherapy with bacillus Calmette-Guérin is the gold standard treatment for patients with carcinoma in situ. Patients with carcinoma in situ refractory to bacillus Calmette-Guérin are candidates for major surgery such as radical cystectomy. We identified the maximum tolerated dose and the recommended dose, and evaluated the safety profile of paclitaxel-hyaluronic acid bioconjugate given by intravesical instillation to patients with carcinoma in situ refractory to bacillus Calmette-Guérin. MATERIALS AND METHODS: A total of 16 patients with carcinoma in situ refractory to bacillus Calmette-Guérin were enrolled in a phase I, open label, single institution study. A minimum of 3 eligible patients were included per dose level. Paclitaxel-hyaluronic acid solution (ONCOFID-P-B™) was administered for 6 consecutive weeks. The primary objective was to identify the maximum tolerated dose and the recommended dose. As secondary objectives the safety profile of ONCOFID-P-B, the pharmacokinetic profile after each instillation and the tumor response were also evaluated. RESULTS: No dose limiting toxicity occurred at any drug level evaluated. The plasma levels of the study drug were always below the lower limit of quantification at all tested doses after each instillation. A total of 11 adverse events were reported by 7 patients and 9 (60%) showed complete treatment response. CONCLUSIONS: Intravesical instillation of ONCOFID-P-B for carcinoma in situ refractory to bacillus Calmette-Guérin showed minimal toxicity and no systemic absorption in the first human intravesical clinical trial to our knowledge. Finally, satisfactory response rates were observed.

In vitro synergistic antibacterial action of certain combinations of gentamicin and essential oils.

The aim of this study was to verify the existence of synergistic antibacterial effect between four essential oils (Aniba rosaeodora, Melaleuca alternifolia, Origanum vulgare, and Pelargonium graveolens) individually combined with the antibacterial drug Gentamicin. We investigated the effectiveness in vitro of the association of essential oil/Gentamicin, against fifteen different strains of Gram positive and Gram negative bacteria. The antibacterial effects of these oils in combination with Gentamicin were evaluated by using the MHB microdilution method, while gas chromatography (GC) and GC/Mass spectrometry were used to analyze the chemical composition of the oils. A synergistic interaction was observed against all tested strains with the associations between the essential oils Aniba rosaeodora/Gentamicin and Pelargonium graveolens/Gentamicin. In particular a very strong synergistic interaction was observed against Acinetobacter baumannii ATCC 19606 (FIC index = 0.11). In contrast, the essential oils Origanum vulgare and Melaleuca alternifolia in association with Gentamicin were less effective on bacterial species growth. In vitro interaction can improve the antimicrobial effectiveness of the Gentamicin and may contribute to reduce its dose correlated to side effects.

Menkes disease.

Menkes disease is caused by mutations in the copper-transporting P(1B)-type ATPase ATP7A. ATP7A has a dual function: it serves to incorporate copper into copper-dependent enzymes, and it maintains intracellular copper levels by removing excess copper from the cytosol. To accomplish both functions, the protein traffics between different cellular locations depending on copper levels. The mechanism for sensing the concentration of copper, for trafficking, as well as the details of the mechanism of copper translocation across the membrane are unknown.