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The pharmacodynamic and pharmacokinetic properties of bioactive peptides can be modulated by introducing conformational constraints such as intramolecular macrocyclizations, which can involve either the backbone and/or side chains. Herein, we aimed at increasing the α-helicity content of temporin L, an isoform of an intriguing class of linear antimicrobial peptides (AMPs), endowed with a wide antimicrobial spectrum, by the employment of diverse side-chain tethering strategies, including lactam, 1,4-substituted [1,2,3]-triazole, hydrocarbon, and disulfide linkers. Our approach resulted in a library of cyclic temporin L analogues that were biologically assessed for their antimicrobial, cytotoxic, and antibiofilm activities, leading to the development of the first-in-class cyclic peptide related to this AMP family. Our results allowed us to expand the knowledge regarding the relationship between the α-helical character of temporin derivatives and their biological activity, paving the way for the development of improved antibiotic cyclic AMP analogues.

Sulfur(VI) fluoride exchange (SuFEx) click chemistry has offered a facile and reliable approach to produce polysulfates and polysulfonates. However, the current SuFEx polymerization methods lack precise control of target molecular weight and dispersity. Herein, we report the first chain-growth SuFEx polycondensation process by exploiting the unique reactivity and selectivity of S–F bonds under SuFEx catalysis. Given the higher reactivity of iminosulfur oxydifluoride versus fluorosulfate, the chain-growth SuFEx polycondensation is realized by using an iminosulfur oxydifluoride-containing compound as the reactive chain initiator and deactivated AB-type aryl silyl ether-fluorosulfates bearing an electron-withdrawing group as monomers. When 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) was utilized as the polymerization catalyst, precise control over the polymer molecular weight and polydispersity was achieved. The resulting polymers possess great thermal stability but are easily degradable under mild acidic and basic conditions., The first chain-growth sulfur(VI) fluoride exchange polycondensation by exploiting the high reactivity of iminosulfur oxydifluorides over fluorosulfates is developed. The resulting polymers possess excellent thermal stability and full degradability under mild acidic and basic conditions.

We present a modified microfluidic open interface (MOI) for the direct coupling of Bio-SPME to a liquid electron ionization-tandem mass spectrometry (LEI-MS/MS) system as a sensitive technique that can directly analyze biological samples without the need for sample cleanup or chromatographic separations as well as without measurable matrix effects (ME). We selected fentanyl as test compound. The method uses a C18 Bio-SPME fiber by direct immersion (DI) in urine and plasma and the subsequent quick desorption (1 min) in a flow-isolated volume (2.5 μL) filled with an internal standard−acetonitrile solution. The sample is then transferred to an EI source of a triple-quadrupole mass spectrometer via a LEI interface at a nanoscale flow rate. The desorption and analysis procedure requires less than 10 min. Up to 150 samples can be analyzed without observing a performance decline, with fentanyl quantitation at microgram-per-liter levels. The method workflow is extremely dependable, relatively fast, sustainable, and leads to reproducible results that enable the high-throughput screening of various biological samples.

In this study, we report the realization of drug-loaded smart magnetic nanocarriers constituted by superparamagnetic iron oxide nanoparticles encapsulated in a dual pH- and temperature-responsive poly (N-vinylcaprolactam-co-acrylic acid) copolymer to achieve highly controlled drug release and localized magnetic hyperthermia. The magnetic core was constituted by flower-like magnetite nanoparticles with a size of 16.4 nm prepared by the polyol approach, with good saturation magnetization and a high specific absorption rate. The core was encapsulated in poly (N-vinylcaprolactam-co-acrylic acid) obtaining magnetic nanocarriers that revealed reversible hydration/dehydration transition at the acidic condition and/or at temperatures above physiological body temperature, which can be triggered by magnetic hyperthermia. The efficacy of the system was proved by loading doxorubicin with very high encapsulation efficiency (>96.0%) at neutral pH. The double pH- and temperature-responsive nature of the magnetic nanocarriers facilitated a burst, almost complete release of the drug at acidic pH under hyperthermia conditions, while a negligible amount of doxorubicin was released at physiological body temperature at neutral pH, confirming that in addition to pH variation, drug release can be improved by hyperthermia treatment. These results suggest this multi-stimuli-sensitive nanoplatform is a promising candidate for remote-controlled drug release in combination with magnetic hyperthermia for cancer treatment.

Herein we present an efficient, column-switching method that relies on a custom-made T-union passive diffusion micromixer to assist water dilution and promote trap solute focusing of a high sample volume dissolved in pure organic solvent using a 0.075 mm i.d. nano-LC column. This method allows injecting 20 μL (or higher) of sample volume, speeding up the analysis time, with a 400-fold increase of the limits of quantitation for selected compounds. Five pesticides in different media were used as model compounds, and the analyses were carried out with a triple quadrupole mass spectrometer equipped with a Liquid Electron Ionization (LEI) LC-MS interface working in multiple reaction monitoring (MRM) mode. The system microfluidics were investigated using COMSOL modeling software. Robustness of the entire system was evaluated using a post-extraction addition soil extracts with limits of detection values spanning from 0.10 to 0.45 µg/L. Reproducible results in terms of peak area, peak shape, and retention times were achieved in soil matrix. Repeatability test on peak area variations were lower than 10%.

Optimization of small-molecule probes or drugs is a synthetically lengthy, challenging, and resource-intensive process. Lack of automation and reliance on skilled medicinal chemists is cumbersome in both academic and industrial settings. Here, we demonstrate a high-throughput hit-to-lead process based on the biocompatible sulfur(VI) fluoride exchange (SuFEx) click chemistry. A high-throughput screening hit benzyl (cyanomethyl)carbamate (Ki = 8 μM) against a bacterial cysteine protease SpeB was modified with a SuFExable iminosulfur oxydifluoride [RN═S(O)F2] motif, rapidly diversified into 460 analogs in overnight reactions, and the products were directly screened to yield drug-like inhibitors with 480-fold higher potency (Ki = 18 nM). We showed that the improved molecule is active in a bacteria-host coculture. Since this SuFEx linkage reaction succeeds on picomole scale for direct screening, we anticipate our methodology can accelerate the development of robust biological probes and drug candidates.

The ability to catalyse a reaction acting on different substrates, known as “broad-specificity” or “multi-specificity”, and to catalyse different reactions at the same active site (“promiscuity”) are common features among the enzymes. These properties appear to go against the concept of extreme specificity of the catalytic action of enzymes and have been re-evaluated in terms of evolution and metabolic adaptation. This paper examines the potential usefulness of a differential inhibitory action in the study of the susceptibility to inhibition of multi-specific or promiscuous enzymes acting on different substrates. Aldose reductase is a multi-specific enzyme that catalyses the reduction of both aldoses and hydrophobic cytotoxic aldehydes and is used here as a concrete case to deal with the differential inhibition approach.

Polysulfonates obtained from SuFEx click reactions can be degraded using DBU as a bifunctional catalyst. A possible mechanism proceeding through the esterification of the R-S(O)-OR′ group is proposed. The degradation process was further utilized to prepare functional polymers such as the AIEgen-ended polymer and a ternary polymer with tunable emission.

This review article traces the history of the use of liquid chromatography coupled with mass spectrometry (LC-MS) using electron ionization (EI) from the first attempts up to the present day. At the time of the first efforts to couple LC to MS, 70 eV EI was the most common ionization technique, typically used in gas chromatography-mass spectrometry (GC-MS) and providing highly reproducible mass spectra that could be collated in libraries. Therefore, it was obvious to transport this dominant approach to the early LC-MS coupling attempts. The use of LC coupled to EI-MS is challenging mainly due to restrictions related to high-vacuum and high-temperature conditions required for the operation of EI and the need to remove the eluent carrying the analyte before entering the ion source. The authors will take readers through a journey of about 50 years, showing how through the succession of different attempts it has been possible to successfully couple LC with EI-MS, which in principle appear to be incompatible.

International audience; 5548 Background: Ovarian cancer is the leading cause of death by gynecological cancer. Complete surgery remains one of the main prognostic factors. Laparoscopic exploration is mandatory to assess surgical resectability at diagnosis or after neoadjuvant chemotherapy. However, there is no clinical or biological marker that can correctly predict resectability and may be able to avoid a second laparoscopic exploration for initially unresectable diseases. Our aim was to assess circulating tumor DNA (ctDNA) value as a predictive non-invasive marker of evolution towards resectability for patients with epithelial ovarian cancer receiving first-line chemotherapy. Methods: We explored in this work one of the secondary objectives of the CIDOC study (NCT03302884). CIDOC is a multicenter prospective study aiming to explore ctDNA value as early marker of disease relapse after first-line treatment for epithelial ovarian cancer. Patients with mucinous histology or early stages not requiring chemotherapy are excluded. Plasma samples are collected at diagnosis, during neoadjuvant chemotherapy, and during follow-up. After DNA extraction, panel-based next generation sequencing is performed on both tumor samples and germline DNA, and somatic mutations of interest are selected for ctDNA monitoring. ctDNA analyses are conducted using droplet digital PCR (BioRad QX200) by measuring the variant allele fraction (VAF) of previously identified mutations. Results: This intermediary analysis has included 47 patients diagnosed between March 2017 and December 2019. Median age was 69 years old (48 – 84). Most of the patients had advanced disease (89.4% stage FIGO III or IV), serous histology (94.8%), and high grade tumor (92.3%). Most of the patients underwent complete interval cytoreductive surgery (76.3% vs 17.4% complete upfront surgery). Most of the tumors had TP53 mutations (85.1%), following by alterations involving DNA repair genes (38.3%). Median cell-free DNA concentration at baseline was 0.38 ng/µL (0 – 12.8). ctDNA was identified in 92.1% of patients at baseline with a median VAF of 1.84% (0 – 42.52%). ctDNA VAF was correlated to the peritoneal dissemination ( p= 0.039) assessed with the peritoneal cancer index. ctDNA clearance after preoperative chemotherapy tended to be correlated to achievement of complete interval surgery for patients receiving neoadjuvant chemotherapy ( p= 0.108). Conclusions: ctDNA may be a promising non-invasive marker to assess peritoneal cancer spreading and to predict surgical resectability after neoadjuvant chemotherapy. If confirmed in larger populations, this may enable to avoid additional surgical explorations for patients who remain ctDNA positive after chemotherapy. Clinical trial information: NCT03302884.

Sulfur (Ⅵ) fluoride exchange (SuFEx) click chemistry has offered a facile and reliable approach to produce polysul-fates and polysulfonates. However, the current SuFEx polymerization methods lack precise control of target molecular weight and dispersity. Herein, we report the first chain-growth SuFEx polycondensation process by exploiting unique reactivity and selectivity of S-F bonds under SuFEx catalysis. Given the higher reactivity of iminosulfur oxydifluoride versus fluorosulfate, the chain-growth SuFEx polycondensation is realized by using an iminosulfur oxydifluoride-containing compound as the reactive chain initiator and deactivated AB-type aryl silyl ether-fluorosulfates bearing an electron-withdrawing group as monomers. When DBU was utilized as the polymerization catalyst, precise control over polymer molecular weight and polydispersity are achieved. The resulting polymers possess great thermal stability but are easily degradable under mild acidic and basic conditions.

Significance Excessive use of antibiotics for patient therapy and livestock results in the selection of pathogenic bacteria resistant to multiple drugs. The spread of multidrug-resistant bacteria into the community is associated with increased morbidity, mortality, and financial burden of health care. We constructed a library of SuFEx-derived arylfluorosulfates that demonstrated potent activities in combating multidrug-resistant bacteria both in vitro and in vivo. Significantly, these arylfluorosulfate analogs show enhanced potency to disrupt bacterial biofilm as well as kill persisters and have a synergistic effect with clinical bactericidal drugs. This study suggests that arylfluorosulfates derived from SuFEx-based modification of phenols could serve as a convenient strategy to search for bactericidal candidates.

Mass spectrometry has been the "gold standard" for drugs of abuse (DoA) analysis for many decades because of the selectivity and sensitivity it affords. Recent progress in all aspects of mass spectrometry has seen significant developments in the field of DoA analysis. Mass spectrometry is particularly well suited to address the rapidly proliferating number of very high potency, novel psychoactive substances that are causing an alarming number of fatalities worldwide. This review surveys advancements in the areas of sample preparation, gas and liquid chromatography-mass spectrometry, as well as the rapidly emerging field of ambient ionization mass spectrometry. We have predominantly targeted literature progress over the past ten years and present our outlook for the future. © 2020 Periodicals, Inc. Mass Spec Rev.

Novel diterpenoids were isolated from the extracts of Fabiana densa var. ramulosa and found to display a selective activity against Gram-positive bacterial strains with negligible cytotoxicity toward human keratinocytes. This study highlighted the role played by the acidic group at C18 of the tetracyclic ent-beyerene scaffold for antibacterial effects and how the length and flexibility of the alkyl chain between the two carbonyl groups are crucial factors to increase the antimicrobial activity of the molecules, supporting the development of natural products from F. densa and their derivatives for treatment of microbial infections.

The rapid development of antimicrobial resistance is pushing the search in the discovering of novel antimicrobial molecules to prevent and treat bacterial infections. Self-assembling antimicrobial peptides, as the lipidated peptides, are a novel and promising class of molecules capable of meeting this need. Based on previous work on Temporin L analogs, several new molecules lipidated at the N- or and the C-terminus were synthesised. Our goal is to improve membrane interactions through finely tuning self-assembly to reduce oligomerisation in aqueous solution and enhance self-assembly in bacterial membranes while reducing toxicity against human cells. The results here reported show that the length of the aliphatic moiety is a key factor to control target cell specificity and the oligomeric state of peptides either in aqueous solution or in a membrane-mimicking environment. The results of this study pave the way for the design of novel molecules with enhanced activities.

BackgroundColistin is a last-resort treatment option for many MDR Gram-negative bacteria. The covalent addition of l-aminoarabinose to the lipid A moiety of LPS is the main colistin resistance mechanism in the human pathogen Pseudomonas aeruginosa.ObjectivesIdentification (by in silico screening of a chemical library) of potential inhibitors of ArnT, which catalyses the last committed step of lipid A aminoarabinosylation, and their validation in vitro as colistin adjuvants.MethodsThe available ArnT crystal structure was used for a docking-based virtual screening of an in-house library of natural products. The resulting putative ArnT inhibitors were tested in growth inhibition assays using a reference colistin-resistant P. aeruginosa strain. The most promising compound was further characterized for its range of activity, specificity and cytotoxicity. Additionally, the effect of the compound on lipid A aminoarabinosylation was verified by MS analyses of lipid A.ResultsA putative ArnT inhibitor (BBN149) was discovered by molecular docking and demonstrated to specifically potentiate colistin activity in colistin-resistant P. aeruginosa isolates, without relevant effect on colistin-susceptible strains. BBN149 also showed adjuvant activity against colistin-resistant Klebsiella pneumoniae and low toxicity to bronchial epithelial cells. Lipid A aminoarabinosylation was reduced in BBN149-treated cells, although only partially.ConclusionsThis study demonstrates that in silico screening targeting ArnT can successfully identify inhibitors of colistin resistance and provides a promising lead compound for the development of colistin adjuvants for the treatment of MDR bacterial infections.

Colistin is a last-resort antibiotic for the treatment of multidrug resistant Gram-negative bacterial infections. Recently, a natural ent-beyerene diterpene was identified as a promising inhibitor of the enzyme responsible for colistin resistance mediated by lipid A aminoarabinosylation in Gram-negative bacteria, namely, ArnT (undecaprenyl phosphate-alpha-4-amino-4-deoxy-l-arabinose arabinosyl transferase). Here, semisynthetic analogues of hit were designed, synthetized, and tested against colistin-resistant Pseudomonas aeruginosa strains including clinical isolates to exploit the versatility of the diterpene scaffold. Microbiological assays coupled with molecular modeling indicated that for a more efficient colistin adjuvant activity, likely resulting from inhibition of the ArnT activity by the selected compounds and therefore from their interaction with the catalytic site of ArnT, an ent-beyerane scaffold is required along with an oxalate-like group at C-18/C-19 or a sugar residue at C-19 to resemble L-Ara4N. The ent-beyerane skeleton is identified for the first time as a privileged scaffold for further cost-effective development of valuable colistin resistance inhibitors.

Dispersive liquid-liquid microextraction with and without ultrasound assistance (DLLME, UA-DLLME) and microextraction with packed sorbent (MEPS) methods for the extraction and determination of eight different benzodiazepines (BDZ) (chlordiazepoxide, flurazepam, bromazepam, oxazepam, lorazepam, clobazam, clonazepam, and flunitrazepam) in three commercial non-alcoholic and light alcoholic beverages were optimized and compared. Benzodiazepines are frequently used for their extensive diffusion and strong numbing effect in drug-facilitated crimes (DFC). The tiny small amount of sample required for DLLME and MEPS extraction makes them very suitable for specimens collected at the crime scene of DFCs. Microextraction techniques are of increasing interest thanks to their accordance to green analytical chemistry (GAC) guidelines providing good recovery values. Ultrasound assistance (UA-DLLME) was used to investigate whether this type of energy can improve the recoveries of the analytes. Analyses of the extracts were performed with reverse-phase capillary high-performance liquid chromatography with UV detection (HPLC − UV), thanks to low environmental impact, robustness, diffusion, and affordability. Recovery percentages at three different concentrations in the three beverages were between 14.30% and 103.28% with intraday and interday RSD lower than ± 2.78%. The same samples were extracted using a MEPS protocol, and the results were compared with those obtained with DLLME. MEPS gave recoveries between 20.90% and 101.88% for all matrices showing a better performance than DLLME at higher concentrations, though lower recoveries were observed with diluted samples.

Extra virgin olive oil (EVOO) is one of the main ingredients of the Mediterranean diet. It is claimed as a functional food for its unique content of health-promoting compounds. Tyrosol (Tyr), Hydroxytyrosol (Htyr), and their phenolic derivatives present in EVOO show beneficial properties, and their identification and quantification, both in their free form and after the hydrolysis of more complex precursors, are important to certify its quality. An alternative method for quantifying free and total Tyr and Htyr in EVOO is presented using an LC–MS interface based on electron ionization (EI), called liquid electron ionization (LEI). This method requires neither sample preparation nor chromatography, the sample is diluted and injected. The selectivity and sensitivity were assessed in multiple reaction monitoring mode (MRM), obtaining confirmation and quantification in actual samples ranging from 5 to 11 mg/Kg for the free forms and from 32 to 80 mg/Kg for their total amount after hydrolysis. Two MS/MS transitions were acquired for both compounds using the Q/q ratios as confirmatory parameters. Standard addition calibration curves demonstrated optimal linearity and negligible matrix effects, allowing a correct quantification even without expensive and difficult to find labeled internal standards. After several weeks of operation, the system’s repeatability was excellent, with an intraday RSD (%) spanning from five to nine and an interday RSD (%) spanning from 9 to 11.

Designed multiple ligands (DMLs), developed to modulate simultaneously a number of selected targets involved in etiopathogenetic mechanisms of a multifactorial disease, such as diabetes mellitus (DM), are considered a promising alternative to combinations of drugs, when monotherapy results to be unsatisfactory. In this work, compounds 1-17 were synthesized and in vitro evaluated as DMLs directed to aldose reductase (AR) and protein tyrosine phosphatase 1B (PTP1B), two key enzymes involved in different events which are critical for the onset and progression of type 2 DM and related pathologies. Out of the tested 4-thiazolidinone derivatives, compounds 12 and 16, which exhibited potent AR inhibitory effects along with interesting inhibition of PTP1B, can be assumed as lead compounds to further optimize and balance the dual inhibitory profile. Moreover, several structural portions were identified as features that could be useful to achieve simultaneous inhibition of both human AR and PTP1B through binding to non-catalytic regions of both target enzymes.

Lung fibrosis results from the synergic interplay between regenerative deficits of the alveolar epithelium and dysregulated mechanisms of repair in response to alveolar and vascular damage, which is followed by progressive fibroblast and myofibroblast proliferation and excessive deposition of the extracellular matrix. The increased parenchymal stiffness of fibrotic lungs significantly affects respiratory mechanics, making the lung more fragile and prone to non-physiological stress during spontaneous breathing and mechanical ventilation. Given their parenchymal inhomogeneity, fibrotic lungs may display an anisotropic response to mechanical stresses with different regional deformations (micro-strain). This behavior is not described by the standard stress-strain curve but follows the mechano-elastic models of "squishy balls", where the elastic limit can be reached due to the excessive deformation of parenchymal areas with normal elasticity that are surrounded by inelastic fibrous tissue or collapsed induration areas, which tend to protrude outside the fibrous ring. Increasing evidence has shown that non-physiological mechanical forces applied to fibrotic lungs with associated abnormal mechanotransduction could favor the progression of pulmonary fibrosis. With this review, we aim to summarize the state of the art on the relation between mechanical forces acting on the lung and biological response in pulmonary fibrosis, with a focus on the progression of damage in the fibrotic lung during spontaneous breathing and assisted ventilatory support.

Persistent infections, such as those provoked by the Gram-negative bacterium Pseudomonas aeruginosa in the lungs of cystic fibrosis (CF) patients, can induce inflammation with lung tissue damage and progressive alteration of respiratory function. Therefore, compounds having both antimicrobial and immunomodulatory activities are certainly of great advantage in fighting infectious diseases and chronic inflammation. We recently demonstrated the potent antipseudomonal efficacy of the antimicrobial peptide (AMP) Esc(1-21) and its diastereomer Esc(1-21)-1c, namely Esc peptides. Here, we confirmed this antimicrobial activity by reporting on the peptides&rsquo, ability to kill P. aeruginosa once internalized into alveolar epithelial cells. Furthermore, by means of enzyme-linked immunosorbent assay and Western blot analyses, we investigated the peptides&rsquo, ability to detoxify the bacterial lipopolysaccharide (LPS) by studying their effects on the secretion of the pro-inflammatory cytokine IL-6 as well as on the expression of cyclooxygenase-2 from macrophages activated by P. aeruginosa LPS. In addition, by a modified scratch assay we showed that both AMPs are able to stimulate the closure of a gap produced in alveolar epithelial cells when cell migration is inhibited by concentrations of Pseudomonas LPS that mimic lung infection conditions, suggesting a peptide-induced airway wound repair. Overall, these results have highlighted the two Esc peptides as valuable candidates for the development of new multifunctional therapeutics for treatment of chronic infectious disease and inflammation, as found in CF patients.

Bacterial biofilms are a serious threat for human health, and the Gram-positive bacterium Staphylococcus aureus is one of the microorganisms that can easily switch from a planktonic to a sessile lifestyle, providing protection from a large variety of adverse environmental conditions. Dormant non-dividing cells with low metabolic activity, named persisters, are tolerant to antibiotic treatment and are the principal cause of recalcitrant and resistant infections, including skin infections. Antimicrobial peptides (AMPs) hold promise as new anti-infective agents to treat such infections. Here for the first time, we investigated the activity of the frog-skin AMP temporin G (TG) against preformed S. aureus biofilm including persisters, as well as its efficacy in combination with tobramycin, in inhibiting S. aureus growth. TG was found to provoke ~50 to 100% reduction of biofilm viability in the concentration range from 12.5 to 100 &micro, M vs ATCC and clinical isolates and to be active against persister cells (about 70&ndash, 80% killing at 50&ndash, 100 &micro, M). Notably, sub-inhibitory concentrations of TG in combination with tobramycin were able to significantly reduce S. aureus growth, potentiating the antibiotic power. No critical cytotoxicity was detected when TG was tested in vitro up to 100 &micro, M against human keratinocytes, confirming its safety profile for the development of a new potential anti-infective drug, especially for treatment of bacterial skin infections.

The loss of genetic fidelity in tissue stem cells is considered a significant cause of human aging and carcinogenesis. Many cellular mechanisms are well accepted for limiting mutations caused by replication errors and DNA damage. However, one mechanism, non-random sister chromatid segregation, remains controversial. This atypical pattern of chromosome segregation is restricted to asymmetrically self-renewing cells. Though first confirmed in murine cells, non-random segregation was originally proposed by Cairns as an important genetic fidelity mechanism in human tissues. We investigated human hepatic stem cells expanded by suppression of asymmetric cell kinetics (SACK) for evidence of non-random sister chromatid segregation. Cell kinetics and time-lapse microscopy analyses established that an ex vivo expanded human hepatic stem cell strain possessed SACK agent-suppressible asymmetric cell kinetics. Complementary DNA strand-labeling experiments revealed that cells in hepatic stem cell cultures segregated sister chromatids non-randomly. The number of cells cosegregating sister chromatids with the oldest &ldquo, immortal DNA strands&rdquo, was greater under conditions that increased asymmetric self-renewal kinetics. Detection of this mechanism in a human tissue stem cell strain increases support for Cairns&rsquo, proposal that non-random sister chromatid segregation operates in human tissue stem cells to limit carcinogenesis.

In this work, the surface of nanostructured fluorine-doped ZnO (nZnO&middot, F) is functionalized with protein A (PrA), and used as a model biomolecule. The chemical procedure is characterized by several analytical techniques such as Fourier Transform Infrared Spectroscopy, water contact angle analysis, and fluorescence microscopy. The surface modification of nZnO&middot, F by binding increasing concentrations of PrA is also investigated by two label-free optical techniques, i.e., the spectroscopic reflectometry and the steady-state photoluminescence. The results are compared with those obtained using undoped nZnO substrates in order to highlight the better performances of nZnO&middot, F due to the fluorine doping. The results of this study pave the way for the design and realization of a ZnO-based nanostructured platform for label-free optical sensing.

Liquid Electron Ionization (LEI), is an innovative liquid chromatography-mass spectrometry (LC-MS) interface that converts liquid HPLC eluent to the gas-phase in a mass spectrometer equipped with an electron ionization (EI) source. LEI extends the electronic spectra libraries access to liquid chromatography, providing a powerful tool in the untargeted approacssh. Negligible matrix effects allow accurate quantitative information. The purpose of this research was to evaluate the main aspects concerning the interfacing process. These fundamental studies were necessary to understand the mechanism of LEI in details, and improve the interfacing process, especially regarding robustness and sensitivity. Hardware components were installed to prevent analytes precipitation, reduce thermal decomposition of sensitive compounds, and to stabilize the nano-flow delivery with different mobile-phase compositions. Particular attention was devoted to insulating the heated vaporization area from the LC part of the system. Experiments were performed to optimize the interface inner capillary dimensions, and other operative parameters, including temperature, gas and liquid flow rates. Test compounds of environmental interest were selected based on molecular weight, thermal stability, volatility, and polarity. Robustness was evaluated with a set of replicated injections and calibration experiments using a soil matrix as a test sample. MRM detection limits in the low-picogram range were obtained for five pesticides belonging to different classes in a soil sample. High-quality electron ionization mass spectra of a mixture of pesticides were also obtained.

Due to their excellent in vitro activity against multidrug resistant bacteria, antimicrobial peptides (AMPs) hold promise for treatment of Pseudomonas aeruginosa lung infections in cystic fibrosis (CF) sufferers. In this work, poly(lactide- co-glycolide) (PLGA) nanoparticles for lung delivery of AMPs deriving from the frog-skin esculentin-1a, namely, Esc(1-21) and Esc(1-21)-1c (Esc peptides), were successfully developed. Improved peptide transport through artificial CF mucus and simulated bacterial extracellular matrix was achieved in vitro. The formulations were effectively delivered through a liquid jet nebulizer already available to patients. Notably, Esc peptide-loaded nanoparticles displayed an improved efficacy in inhibiting P. aeruginosa growth in vitro and in vivo in the long term. A single intratracheal administration of Esc peptide-loaded nanoparticles in a mouse model of P. aeruginosa lung infection resulted in a 3-log reduction of pulmonary bacterial burden up to 36 h. Overall, results unravel the potential of PLGA nanoparticles as a reliable delivery system of AMPs to lungs.

Buprenorphine and its metabolites are routinely monitored to assess patient compliance with drug detoxification programs or as pain killers. A rapid method for the simultaneous analysis of buprenorphine, norbuprenorphine, and glucuronides in urine using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed. Urine samples were diluted in water containing formic acid 0.1% and directly injected into the UHPLC-MS/MS system without any sample pretreatment. Quality control (QC) samples, prepared using 20 different urine matrices, fortified at 3 concentration levels, were quantified using four deuterated internal standards. The accuracy values obtained spanned from 90 to 114% with repeatability lower than 10% also in the inter-day between batch experiments. Matrix effects (ME), evaluated before correction with internal standards using Matuszewski procedure, mainly affected the analysis of buprenorphine glucuronide. The use of deuterated internal standards (IS) for each analyte was necessary to compensate for ME and was essential in the determination of glucuronides. The method was applied to 30 real urine samples from patients under a detoxification therapy. Duplicate analyses were performed with the presented method and compared with another method which involves a standard hydrolysis procedure. Real sample results were compared showing a good performance agreement, with differences between the two methods lower than ±20% in the quantification results.

Background:\ud State-of-the art therapy for recurrent ovarian cancer suitable for platinum-based re-treatment includes bevacizumab-containing combinations (eg, bevacizumab combined with carboplatin–paclitaxel or carboplatin–gemcitabine) or the most active non-bevacizumab regimen: carboplatin–pegylated liposomal doxorubicin. The aim of this head-to-head trial was to compare a standard bevacizumab-containing regimen versus carboplatin–pegylated liposomal doxorubicin combined with bevacizumab.\ud Methods:\ud This multicentre, open-label, randomised, phase 3 trial, was done in 159 academic centres in Germany, France, Australia, Austria, and the UK. Eligible patients (aged ≥18 years) had histologically confirmed epithelial ovarian, primary peritoneal, or fallopian tube carcinoma with first disease recurrence more than 6 months after first-line platinum-based chemotherapy, and an Eastern Cooperative Oncology Group performance status of 0–2. Patients were stratified by platinum-free interval, residual tumour, previous antiangiogenic therapy, and study group language, and were centrally randomly assigned 1:1 using randomly permuted blocks of size two, four, or six to receive six intravenous cycles of bevacizumab (15 mg/kg, day 1) plus carboplatin (area under the concentration curve [AUC] 4, day 1) plus gemcitabine (1000 mg/m 2, days 1 and 8) every 3 weeks or six cycles of bevacizumab (10 mg/kg, days 1 and 15) plus carboplatin (AUC 5, day 1) plus pegylated liposomal doxorubicin (30 mg/m 2, day 1) every 4 weeks, both followed by maintenance bevacizumab (15 mg/kg every 3 weeks in both groups) until disease progression or unacceptable toxicity. There was no masking in this open-label trial. The primary endpoint was investigator-assessed progression-free survival according to Response Evaluation Criteria in Solid Tumors version 1.1. Efficacy data were analysed in the intention-to-treat population. Safety was analysed in all patients who received at least one dose of study drug. This completed study is registered with ClinicalTrials.gov, NCT01837251.\ud Findings:\ud Between Aug 1, 2013, and July 31, 2015, 682 eligible patients were enrolled, of whom 345 were randomly assigned to receive carboplatin–pegylated liposomal doxorubicin–bevacizumab (experimental group) and 337 were randomly assigned to receive carboplatin–gemcitabine–bevacizumab (standard group). Median follow-up for progression-free survival at data cutoff (July 10, 2018) was 12·4 months (IQR 8·3–21·7) in the experimental group and 11·3 months (8·0–18·4) in the standard group. Median progression-free survival was 13·3 months (95% CI 11·7–14·2) in the experimental group versus 11·6 months (11·0–12·7) in the standard group (hazard ratio 0·81, 95% CI 0·68–0·96; p=0·012). The most common grade 3 or 4 adverse events were hypertension (88 [27%] of 332 patients in the experimental group vs 67 [20%] of 329 patients in the standard group) and neutropenia (40 [12%] vs 73 [22%]). Serious adverse events occurred in 33 (10%) of 332 patients in the experimental group and 28 (9%) of 329 in the standard group. Treatment-related deaths occurred in one patient in the experimental group (

Antimicrobial Peptides (AMPs) are the key effectors of the innate immunity and represent promising molecules for the development of new antibacterial drugs. However, to achieve this goal, some problems need to be overcome: (i) the cytotoxic effects at high concentrations; (ii) the poor biostability and (iii) the difficulty in reaching the target site. Frog skin is one of the richest natural storehouses of AMPs, and over the years, many peptides have been isolated from it, characterized and classified into several families encompassing temporins, brevinins, nigrocins and esculentins. In this review, we summarized how the isolation/characterization of peptides belonging to the esculentin-1 family drove us to the design of an analogue, i.e. esculentin-1a(1-21)NH2, with a powerful antimicrobial action and immunomodulatory properties. The peptide had a wide spectrum of activity, especially against the opportunistic Gram-negative bacterium Pseudomonas aeruginosa. We described the structural features and the in vitro/in vivo biological characterization of this peptide as well as the strategies used to improve its biological properties. Among them: (i) the design of a diastereomer carrying Damino acids in order to reduce the peptide’s cytotoxicity and improve its half-life; (ii) the covalent conjugation of the peptide to gold nanoparticles or its encapsulation into poly(lactide- co-glycolide) nanoparticles; and (iii) the peptide immobilization to biomedical devices (such as silicon hydrogel contact lenses) to obtain an antibacterial surface able to reduce microbial growth and attachment. Summing up the best results obtained so far, this review traces all the steps that led these frog-skin AMPs to the direction of peptide-based drugs for clinical use.

In the past few years, a huge research effort has been performed in order to investigate hydrogen and its possible energy use. Hydrogen is considered extremely promising as a reliable energy carrier for replacing in the next future fossil fuels, with the aim of facing the global warming issues. One of the main advantages of hydrogen lies in its production because it can be produced by a plurality of energy sources. In particular, photovoltaic (PV) energy may be used for producing hydrogen employing water electrolysis. The most common electrolysis technologies are: (i) alkaline electrolysis; (ii) proton exchange membrane electrolysis; (iii) solid oxide electrolysis; and (iv) anion exchange membrane electrolysis. Alkali electrolysis is the most mature, studied, and used electrolysis method. Proton exchange membrane electrolysis cells are competing with alkali cells for higher hydrogen purity, higher current density, and a greater operating range. Solid oxide electrolysis, developed for overcoming the disadvantages of proton exchange membrane and alkali electrolysis, is still in the precommercial stage, whereas anion exchange membrane electrolysis is at the laboratory stage. The PV-driven electrolysis process is considered to be a major strategy for the fully renewable production of hydrogen. The major limit of this technology is related to the mismatching between the I-V curve of the PV panel and the electrolyzer. Alkali PV hydrogen production may be presently considered as at commercial status, due to the maturity of the alkaline electrolysis technology. But this technology is limited by the fact that the alkali stack is not able to follow rapid variations of input current densities. Conversely, proton membrane exchange electrolyzers exhibit optimal performance at partial load, suitably managing the fluctuations of the PV production. This chapter focuses on the integration of existing electrolysis techniques with PV plants. Complex hybrid polygenerative systems, also including PV panels and electrolyzers, are out of the scope of this chapter.

Background. Recently, antimicrobial peptides (AMPs) have been investigated for their use in cancer therapy. They have been reported to selectively target and kill cancer cells whilst leaving normal healthy cells unaffected. Certain Anura AMPs have expressed selective cytotoxicity against tumour cells. Aim. To test the potential of Anura AMPs bombinin H2, bombinin H4, temporin A, and temporin L for use as therapeutic agents for non-small cell lung carcinoma (NSCLC). Methods. Cytotoxic effects on NSCLC cell lines A549 and Calu-3 and normal epithelial cell line Beas-2B were tested using the CellTox Green Cytotoxicity Assay. Their haemolytic effects on human erythrocytes were also tested for their clinical relevance. Cell membrane profiling, using MALDI-TOF, was performed to ascertain if membrane characteristics of the NSCLC and Beas-2B cell lines may contribute to the AMPs mode of action. Results. Bombinin H4 (100–1.5 μM, p<0.05) and temporin A (100–50 μM, p<0.05) showed selective cytotoxicity towards the NSCLC cell lines. Furthermore, they exhibited low levels of haemolytic activity (bombinin H4, 0.061%; temporin A, 0.874%) comparable to untreated cells. Cell membrane profiling showed the phospholipid composition of normal epithelial cell line Beas-2B to be divergent from the cancerous cell lines. However, there was an overlap in the phospholipid profiles of the NSCLC cell lines supporting the hypothesis that the AMPs may have a selective affinity via the membrane composition of cancerous cell lines. Conclusion. These results suggest that bombinin H4 and temporin A show potential for application in lung cancer therapies. Further in vitro and in vivo studies are required to develop a greater understanding of their use as anticancer agents.

The airway epithelium is seriously damaged upon pulmonary Pseudomonas aeruginosa infection, especially in cystic fibrosis (CF) sufferers. Therefore, the discovery of novel anti-infective agents accelerating healing of infected injured tissues is crucial. The antipseudomonal peptides esculentin-1a(1–21)NH2 and its diastereomer Esc(1–21)-1c (Esc peptides) hold promise in this respect. In fact, they stimulate airway epithelial wound repair, but no mechanistic insights are available. Here we demonstrated that this process occurs through promotion of cell migration by an indirect activation of epidermal growth factor receptor mediated by metalloproteinases. Furthermore, we showed an increased expression of metalloproteinase 9, at both gene and protein levels, in peptide-treated bronchial epithelial cells with a functional or mutated form of CF transmembrane conductance regulator. In addition, the two peptides counteracted the inhibitory effect of Pseudomonas lipopolysaccharide (mimicking an infection condition) on the wound healing activity of the airway epithelium, and they enhanced the production of interleukin-8 from both types of cells. Finally, no immunogenicity was discovered for Esc peptides, suggesting their potential safety for clinical usage. Besides representing a step forward in understanding the molecular mechanism underlying the peptide-induced wound healing activity, these studies have contributed to highlight Esc peptides as valuable therapeutics with multiple functions.