Febrile sepsis: first report of human disease due to Paenibacillus silvae.

Introduction: Paenibacillus species are saprophytes widely distributed in nature and rarely associated with overt human infection. Most cases have been described in people with important comorbidities and/or immunodepression. We report here what is, to the best of our knowledge, the first documented case of human disease due to Paenibacillus silvae , so far considered an exclusively environmental micro-organism. Case presentation: A 57-year-old female patient was referred to our Unit after a 2 month history of remittent fever. Upon admission, a septic state and bacteraemia were revealed; P. sylvae was identified by 16S rRNA gene amplification and sequencing with matrix-assisted laser desorption/ionization-time of flight MS. The patient became afebrile after 9 days of antibiotic treatment and was completely cured after a 2 week regimen with intravenous amoxicillin-clavulanate plus oral doxycycline. Conclusion: The patient did not report any previous episode of infection. Most of the well-known risk factors to Paenibacillus bacteraemia, i.e. invasive procedures, use of intravenous drugs and foreign bodies, could be excluded, although her immune system was probably impaired due to obesity and heavy smoking. We suggest that the isolation of bacteria belonging to the genus Paenibacillus should not be disregarded, since there is accumulating evidence that these organisms may cause disease even in immunocompetent subjects.

Activated Porous Carbon Fiber: New Adsorbent for Sampling and Analysis by Thermal Desorption of Siloxanes in Biogas and Biomethane.

The growing global energy demand requires the continuous development and optimization of the production of alternative energy sources. According to the circular economy approach, waste conversion into biogas and biomethane represent an interesting energy source. The input into the distribution network and energy conversion systems of biomethane requires quality monitoring and the use of cleaning up systems. Therefore, there is a need to constantly invest in the development of sampling and analysis systems that save time, costs, and materials. The purpose of this study was to use activated porous carbon fiber (APCF), an extremely versatile material for sampling and analysis by thermal desorption, to show the advantages it has over the adsorbents traditionally used for siloxane monitoring. Siloxanes are among the contaminating compounds that are mainly present in biogas and biomethane, and if not removed sufficiently, they endanger the quality and use of the gas. These are highly harmful compounds since during combustion, they produce quartz particles that are abrasive to the surfaces of the materials involved in the energy production process. In addition, siloxanes directly hinder the energy properties of biomethane during combustion, due to their radical scavenger properties. In this work, the efficiency of APCF tube was evaluated by comparing it with common multilayer tube thought sampling and analyzing siloxanes in lab scale and in real scale (biogas plant). Thermal desorption analysis coupled with GC-MS for the determination of siloxanes showed that the use of APCF allows to obtain better performance. This allows to deduce that APCF is an innovative material for the establishment of a better sampling and analysis method than the current ones, enabling better results to be achieved in the process of monitoring fuel quality in biomethane production and storage facilities.

Discovery of novel SARS-CoV-2 inhibitors targeting the main protease Mpro by virtual screenings and hit optimization.

Two years after its emergence, SARS-CoV-2 still represents a serious and global threat to human health. Antiviral drug development usually takes a long time and, to increase the chances of success, chemical variability of hit compounds represents a valuable source for the discovery of new antivirals. In this work, we applied a platform of variably oriented virtual screening campaigns to seek for novel chemical scaffolds for SARS-CoV-2 main protease (Mpro) inhibitors. The study on the resulting 30 best hits led to the identification of a series of structurally unrelated Mpro inhibitors. Some of them exhibited antiviral activity in the low micromolar range against SARS-CoV-2 and other human coronaviruses (HCoVs) in different cell lines. Time-of-addition experiments demonstrated an antiviral effect during the viral replication cycle at a time frame consistent with the inhibition of SARS-CoV-2 Mpro activity. As a proof-of-concept, to validate the pharmaceutical potential of the selected hits against SARS-CoV-2, we rationally optimized one of the hit compounds and obtained two potent SARS-CoV-2 inhibitors with increased activity against Mpro both in vitro and in a cellular context, as well as against SARS-CoV-2 replication in infected cells. This study significantly contributes to the expansion of the chemical variability of SARS-CoV-2 Mpro inhibitors and provides new scaffolds to be exploited for pan-coronavirus antiviral drug development.

Evaluation of the concentration of the toxic 2,3,6,7-tetrachlorobiphenylene in air after an electrical material fire.

It is known that when fires or explosions involve electrical systems, along with PCDDs and PCDFs, polychlorinated biphenylenes (PCBPs) are also produced. These chlorinated tricyclic aromatic pollutants were noticed in fire rubbles and after the World Trade Center destruction. However, the analytical difficulties in developing an efficient method have limited the knowledge of their environmental distribution. In light of the equipotency of 2,3,6,7-TeCBP and 2,3,7,8-TeCDD, PCBPs call for more accurate investigations. In this paper, for the first time, the level and persistence of 2,3,6,7-TeCBP have been investigated in air samples (both indoor and outdoor) after a fire broke out in an industrial building. GC-MS/MS analysis revealed that 2,3,6,7-TeCBP concentrations after the fire (3046 fg/m3 at the "epicentre") were remarkably higher than that of the 2,3,7,8-TeCDD. Moreover, the monitoring for over two years has demonstrated the persistent nature of this compound. 2,3,6,7-TeCBP was also analyzed in two different ambient air scenario: industrial and periurban areas and in both cases its concentrations were no matter of concern, confirming the correlation of 2,3,6,7-TeCBP with fire episodes. Collectively, 2,3,6,7-TeCBP, because of its toxicity, concentration and persistence, is a crucial compound in the evaluation of the health effects correlated with fires of electrical systems.

Validation studies on activated carbon fiber passive sampler for PCDD/Fs and PCBs in water.

The toxicity of polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) is well known, and for this reason studying and monitoring these chemicals is fundamental. Activated carbon fibers (ACFs) are made of an adsorbent material widely used in the industrial field for the removal of micropollutants. The first step in this work was to perform a physico-chemical characterization of the adsorbent, focused on the analytical use of it. In particular, its specific surface area was defined around 2500 m2/g consisting in a homogeneous microporosity distribution and the characterization of ACF surface functional groups pointed out a balance between basic and acidic group. The validity of using the ACF as solid phase extraction and as passive sampler for PCDD/Fs and PCBs in water, has been evaluated by the percentage recovery (R %) of 13C12-labeled standards of PCDD/Fs and PCBs added in a known volume of water. The results were compared to the R% of Liquid-Liquid Extraction which showed a better reproducibility of the results and the proposed method satisfy completely the requirements of US EPA reference methods.

Discovering New Casein Kinase 1d Inhibitors with an Innovative Molecular Dynamics Enabled Virtual Screening Workflow.

The value of including protein flexibility in structure-based drug design (SBDD) is widely documented, and currently, molecular dynamics (MD) simulations represent a powerful tool to investigate protein dynamics. Yet, the inclusion of MD-derived information in pre-existing SBDD workflows is still far from trivial. We recently published an integrated MD-FLAP (Fingerprints for Ligands and Proteins) approach combining MD, clustering and Linear Discriminant Analysis (LDA) for enhancing accuracy, efficacy, and for protein conformational selection in virtual screening (VS) campaigns. Here we prospectively applied the MD-FLAP workflow to discover novel chemotypes inhibiting the Casein Kinase 1 delta (CSNK1D) enzyme. We first obtained a VS model able to separate active from inactive compounds, with a global AUC of 0.9 and a partial ROC enrichment at 0.5% of 0.18, and use it to mine the internal Pfizer screening database. Seven active molecules sharing a phenyl-indazole scaffold, not yet reported among CSNK1D inhibitors, were found. The most potent inhibitor showed an IC50 of 134 nM.

Air pollution survey across the western Mediterranean Sea: overview on oxygenated volatile hydrocarbons (OVOCs) and other gaseous pollutants.

Despite the Mediterranean Sea basin is among the most sensitive areas over the world for climate change and air quality issues, it still remains less studied than the oceanic regions. The domain investigated by the research ship Minerva Uno cruise in Summer 2015 was the Tyrrhenian Sea. An overview on the marine boundary layer (MBL) concentration levels of carbonyl compounds, ozone (O3), and sulfur dioxide (SO2) is reported. The north-western Tyrrhenian Sea samples showed a statistically significant difference in acetone and SO2 concentrations when compared to the south-eastern ones. Acetone and SO2 values were higher in the southern part of the basin; presumably, a blend of natural (including volcanism) and anthropogenic (shipping) sources caused this difference. The mean acetone concentration reached 5.4 µg/m3; formaldehyde and acetaldehyde means were equal to 1.1 µg/m3 and 0.38 µg/m3, respectively. Maximums of 3.0 µg/m3 for formaldehyde and 1.0 µg/m3 for acetaldehyde were detected along the route from Civitavecchia to Fiumicino. These two compounds were also present at levels above the average in proximity of petrol-refining plants on the coast; in fact, formaldehyde reached 1.56 µg/m3 and 1.60 µg/m3, respectively, near Milazzo and Augusta harbors; meanwhile, acetaldehyde was as high as 0.75 µg/m3 at both sites. The levels of formaldehyde agreed with previously reported measurements over Mediterranean Sea and elsewhere; besides, a day/night trend was observed, confirming the importance of photochemical formation for this pollutant. According to this study, Mediterranean Sea basin, which is a closed sea, was confirmed to suffer a high anthropic pressure impacting with diffuse emissions, while natural contribution to pollution could come from volcanic activity, particularly in the south-eastern Tyrrhenian Sea region.

First virtual screening and experimental validation of inhibitors targeting GES-5 carbapenemase.

The worldwide spread of beta-lactamases with hydrolytic activity extended to last resort carbapenems is aggravating the antibiotic resistance problem and endangers the successful antimicrobial treatment of clinically relevant pathogens. As recently highlighted by the World Health Organization, new strategies to contain antimicrobial resistance are urgently needed. Class A carbapenemases include members of the KPC, GES and SFC families. These enzymes have the ability to hydrolyse penicillins, cephalosporins and carbapenems, while also being less susceptible to available beta-lactam inhibitors, such as clavulanic acid. The KPC family is the most prevalent. It is mostly found on plasmids in Klebsiella pneumoniae, meaning that great amounts of attention, in terms of inhibitor design and structural biology, have been dedicated to it, whereas no efforts have yet been dedicated to GES-type enzymes, despite their ability to rapidly and horizontally disseminate. We herein report the first in silico screening against GES-5, which is the most dangerous GES-type beta-lactamase, using a library of 800K commercially available candidates that all share drug-like properties, such as their MW, logP, rotatable bonds and HBA/HBD atoms. The best screening results were filtered to enrich the number of different chemotypes, and then submitted to molecular docking. The 34 most promising candidates were selected for in vitro validation in biochemical assays against recombinant GES-5. Six hits acted as inhibitors, in the high micromolar range, towards GES-5 and led to the identification of the first, novel chemotypes with inhibitory activity against this clinically relevant carbapenemase.

Human dopamine transporter: the first implementation of a combined in silico/in vitro approach revealing the substrate and inhibitor specificities.

Parkinson's disease (PD) is characterized by the loss of dopamine-generating neurons in the substantia nigra and corpus striatum. Current treatments alleviate PD symptoms rather than exerting neuroprotective effect on dopaminergic neurons. New drugs targeting the dopaminergic neurons by specific uptake through the human dopamine transporter (hDAT) could represent a viable strategy for establishing selective neuroprotection. Molecules able to increase the bioactive amount of extracellular dopamine, thereby enhancing and compensating a loss of dopaminergic neurotransmission, and to exert neuroprotective response because of their accumulation in the cytoplasm, are required. By means of homology modeling, molecular docking, and molecular dynamics simulations, we have generated 3D structure models of hDAT in complex with substrate and inhibitors. Our results clearly reveal differences in binding affinity of these compounds to the hDAT in the open and closed conformations, critical for future drug design. The established in silico approach allowed the identification of promising substrate compounds that were subsequently analyzed for their efficiency in inhibiting hDAT-dependent fluorescent substrate uptake, through in vitro live cell imaging experiments. Taken together, our work presents the first implementation of a combined in silico/in vitro approach enabling the selection of promising dopaminergic neuron-specific substrates.

From Experiments to a Fast Easy-to-Use Computational Methodology to Predict Human Aldehyde Oxidase Selectivity and Metabolic Reactions.

Aldehyde oxidase (AOX) is a molibdo-flavoenzyme that has raised great interest in recent years, since its contribution in xenobiotic metabolism has not always been identified before clinical trials, with consequent negative effects on the fate of new potential drugs. The fundamental role of AOX in metabolizing xenobiotics is also due to the attempt of medicinal chemists to stabilize candidates toward cytochrome P450 activity, which increases the risk for new compounds to be susceptible to AOX nucleophile attack. Therefore, novel strategies to predict the potential liability of new entities toward the AOX enzyme are urgently needed to increase effectiveness, reduce costs, and prioritize experimental studies. In the present work, we present the most up-to-date computational method to predict liability toward human AOX (hAOX), for applications in drug design and pharmacokinetic optimization. The method was developed using a large data set of homogeneous experimental data, which is also disclosed as Supporting Information .

Analysis of polychlorodibenzo-p-dioxins and polychlorodibenzofurans in stationary source emissions in GC-MS/MS using hydrogen as the carrier gas.

The European and American methods for the determination of polychlorodibenzo-p-dioxins and polychlorodibenzofurans in stationary source emissions require a high-resolution mass spectrometer detector. At the same time, all of the 17 toxic 2,3,7,8-chlorine-substituted isomers have to be separated by an appropriate chromatographic method. The resulting analysis has long runtimes and sometimes a double-column run is needed, which results in a huge waste of time, money and manual labor. To obtain a fast and cost-effective but still reliable analytical system, a single-column gas chromatography with hydrogen as carrier gas and tandem mass spectrometry detection is proposed. The use of hydrogen as the carrier gas is a good choice to shorten runtimes and improve the chromatographic resolution, while tandem mass spectrometry is a well-known alternative for this analysis. All the chromatographic and mass spectrometric parameters fill the requirements of the reference methods with a 35% reduction of runtimes. The accuracy is tested with three fly ash samples of a proficiency interlaboratory test. A good correlation between the results is obtained (R2  = 0.992, slope = 0.9675), and no coelutions are noted. The system robustness is tested during 5 years of constant use and the maximal relative standard deviation of the relative response factor is 18.8%.

A Pipeline To Enhance Ligand Virtual Screening: Integrating Molecular Dynamics and Fingerprints for Ligand and Proteins.

The importance of taking into account protein flexibility in drug design and virtual ligand screening (VS) has been widely debated in the literature, and molecular dynamics (MD) has been recognized as one of the most powerful tools for investigating intrinsic protein dynamics. Nevertheless, deciphering the amount of information hidden in MD simulations and recognizing a significant minimal set of states to be used in virtual screening experiments can be quite complicated. Here we present an integrated MD-FLAP (molecular dynamics-fingerprints for ligand and proteins) approach, comprising a pipeline of molecular dynamics, clustering and linear discriminant analysis, for enhancing accuracy and efficacy in VS campaigns. We first extracted a limited number of representative structures from tens of nanoseconds of MD trajectories by means of the k-medoids clustering algorithm as implemented in the BiKi Life Science Suite ( http://www.bikitech.com [accessed July 21, 2015]). Then, instead of applying arbitrary selection criteria, that is, RMSD, pharmacophore properties, or enrichment performances, we allowed the linear discriminant analysis algorithm implemented in FLAP ( http://www.moldiscovery.com [accessed July 21, 2015]) to automatically choose the best performing conformational states among medoids and X-ray structures. Retrospective virtual screenings confirmed that ensemble receptor protocols outperform single rigid receptor approaches, proved that computationally generated conformations comprise the same quantity/quality of information included in X-ray structures, and pointed to the MD-FLAP approach as a valuable tool for improving VS performances.

Assessment of nitrous oxide emission from cement plants: real data measured with both Fourier transform infrared and nondispersive infrared techniques.

Nitrous oxide (N2O) is the third most important greenhouse gas after carbon dioxide and methane, and contributes about 6% to the greenhouse effect. Nitrous oxide is a minor component of the atmosphere, and it is a thousand times less than carbon dioxide (CO2). Nevertheless, it is much more potent than CO2 and methane, owing to its long stay in the atmosphere of approximately 120 yr and the high global warmingpotential (GWP) of298 times that of CO2. Although greenhouse gases are natural in the atmosphere, human activities have changed the atmospheric concentrations. Most of the values of emission of nitrous oxide are still obtained by means ofemission factors and not actually measured; the lack ofreal data may result in an underestimation ofcurrent emissions. The emission factors used for the calculation of N2O can be obtained from the "Guidelines for the implementation of the national inventory of emissions" of the Intergovernmental Panel on Climate Change, which refer to all nations for the realization of their inventory. This study will present real data, measured in several Italian cement plants with different characteristics. The work also shows a comparison between N2O concentration measured with in situ-Fourier transform IR (FTIR) and the reference method EN ISO 21258 based on nondispersive IR (NDIR), in order to investigate the interfering compounds in the measurement with NDIR.

Targeting cystalysin, a virulence factor of treponema denticola-supported periodontitis.

Cystalysin from Treponema denticola is a pyridoxal 5'-phosphate dependent lyase that catalyzes the formation of pyruvate, ammonia, and sulfide from cysteine. It is a virulence factor in adult periodontitis because its reaction contributes to hemolysis, which sustains the pathogen. Therefore, it was proposed as a potential antimicrobial target. To identify specific inhibitors by structure-based in silico methods, we first validated the crystal structure of cystalysin as a reliable starting point for the design of ligands. By using single-crystal absorption microspectrophotometry, we found that the enzyme in the crystalline state, with respect to that in solution, exhibits: 1) the same absorption spectra for the catalytic intermediates, 2) a close pKa value for the residue controlling the keto enamine ionization, and 3) similar reactivity with glycine, L-serine, L-methionine, and the nonspecific irreversible inhibitor aminoethoxyvinylglycine. Next, we screened in silico a library of 9357 compounds with the Fingerprints for Ligands and Proteins (FLAP) software, by using the three-dimensional structure of cystalysin as a template. From the library, 17 compounds were selected and experimentally evaluated by enzyme assays and spectroscopic methods. Two compounds were found to competitively inhibit recombinant T. denticola cystalysin, with inhibition constant (Ki ) values of 25 and 37 µM. One of them exhibited a minimum inhibitory concentration (MIC) value of 64 µg mL(-1) on Moraxella catarrhalis ATCC 23246, which proves its ability to cross bacterial membranes.

Determination of free fatty acids in beer wort.

The importance of free fatty acids (FFAs) in wort has been known for a long time because of their influence on beer quality and yeast metabolism. Lipids have a beneficial effect on yeast growth during fermentation as well as negative effects on beer quality. Lipids content of beer affects the ability to form a stable head of foam and plays an important role in beer staling. Moreover, the ratio of unsaturated and saturated fatty acids seems to be related to gushing problems. A novel, simple, and reliable procedure for quantitative analysis of FFAs in wort was developed and validated. The determination of FFAs in wort was achieved via liquid-liquid cartridge extraction, purification of FFA fraction by solid phase extraction, boron trifluoride in methanol methylation, and injection into GC-FID system. The proposed method has high accuracy (<0.3%, expressed as the bias), high precision (<1.2%, RSD), and recoveries ranging from 74% to 98%. The method was tested on two different wort samples (9° and 12° Plato).

Isozyme-specific ligands for O-acetylserine sulfhydrylase, a novel antibiotic target.

The last step of cysteine biosynthesis in bacteria and plants is catalyzed by O-acetylserine sulfhydrylase. In bacteria, two isozymes, O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, have been identified that share similar binding sites, although the respective specific functions are still debated. O-acetylserine sulfhydrylase plays a key role in the adaptation of bacteria to the host environment, in the defense mechanisms to oxidative stress and in antibiotic resistance. Because mammals synthesize cysteine from methionine and lack O-acetylserine sulfhydrylase, the enzyme is a potential target for antimicrobials. With this aim, we first identified potential inhibitors of the two isozymes via a ligand- and structure-based in silico screening of a subset of the ZINC library using FLAP. The binding affinities of the most promising candidates were measured in vitro on purified O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B from Salmonella typhimurium by a direct method that exploits the change in the cofactor fluorescence. Two molecules were identified with dissociation constants of 3.7 and 33 µM for O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, respectively. Because GRID analysis of the two isoenzymes indicates the presence of a few common pharmacophoric features, cross binding titrations were carried out. It was found that the best binder for O-acetylserine sulfhydrylase-B exhibits a dissociation constant of 29 µM for O-acetylserine sulfhydrylase-A, thus displaying a limited selectivity, whereas the best binder for O-acetylserine sulfhydrylase-A exhibits a dissociation constant of 50 µM for O-acetylserine sulfhydrylase-B and is thus 8-fold selective towards the former isozyme. Therefore, isoform-specific and isoform-independent ligands allow to either selectively target the isozyme that predominantly supports bacteria during infection and long-term survival or to completely block bacterial cysteine biosynthesis.

Exposition and reactivity optimization to predict sites of metabolism in chemicals.

Chemical modifications of drugs induced by phase I biotransformations significantly affect their pharmacokinetic properties. Because the metabolites produced can themselves have a pharmacological effect and an intrinsic toxicity, medicinal chemists need to accurately predict the sites of metabolism (SoM) of drugs as early as possible. However, site of metabolism prediction is rarely accompanied by a prediction of the relative abundance of the various metabolites. Such a prediction would be a great help in the study of drug­ drug interactions and in the process of reducing the toxicity of potential drug candidates. The aim of this paper is to present recent developments in the prediction of xenobiotic metabolism and to use concrete examples to explain the computational mechanism employed.

Extrapulmonary mycobacterial infections in a cohort of HIV-positive patients: ultrasound experience from Vicenza, Italy.

PURPOSE: Extrapulmonary tuberculosis (EPTB) is frequently seen in human immunodeficiency virus (HIV)-infected individuals in Sub-Saharan Africa and recent work has shown point-of-care (POC) ultrasound to be a diagnostic aid in the resource-limited, highly endemic setting. Its role in industrialized countries, however, has rarely been studied. With international migration, EPTB is increasingly seen in European hospitals. This study reports ultrasound findings and discusses the diagnostic relevance of EPTB in an industrialized country setting. METHODS: In this retrospective study, we describe a cohort of 27 patients with a predominantly immigrant background diagnosed with HIV and EPTB in Northern Italy and evaluate the role of ultrasound in their clinical management. All inpatient files of HIV-positive individuals admitted to our hospital with culture-proven diagnosis of EPTB were reviewed, along with chest X-rays and ultrasound studies. The outcome and results of long-term follow-up were extracted. RESULTS: A total of 243 HIV-positive inpatients were identified between January 2005 and November 2011. Twenty-seven of the patients [11.1 %, 95 % confidence interval (CI) 7.4-15.7] were diagnosed with EPTB. Ultrasound showed a typical pattern of enlarged abdominal lymph nodes and focal lesions in the spleen and liver in 22 patients (81.5 %, 95 % CI 7.4-15.7) and, thus, helped to raise the suspicion of mycobacterial infection. CONCLUSION: As disseminated mycobacterial infections in HIV-positive patients can be treated effectively if diagnosed early, and typical sonographic findings are seen in the majority of these patients, we suggest that POC ultrasound should be integrated in diagnostic and screening algorithms for EPTB in developed countries.

Inhibitor of ovarian cancer cells growth by virtual screening: a new thiazole derivative targeting human thymidylate synthase.

Human thymidylate synthase (hTS) was targeted through a virtual screening approach. The most optimal inhibitor identified, 2-{4-hydroxy-2-[(2-hydroxybenzylidene)hydrazono]-2,5-dihydrothiazol-5-yl}-N-(3-trifluoromethylphenyl)acetamide (5), showed a mixed-type inhibition pattern, with a K(i) of 1.3 µM and activity against ovarian cancer cell lines with the same potency as cisplatin. X-ray studies revealed that it binds the inactive enzyme conformation. This study is the first example of a nonpeptidic inhibitor that binds the inactive hTS and exhibits anticancer activity against ovarian cancer cells.

Fatal Myocarditis in Course of Plasmodium falciparum Infection: Case Report and Review of Cardiac Complications in Malaria.

We describe a fatal case of imported malaria where the sole finding revealed at the postmortem evaluation was an acute lymphocytic myocarditis with myocardiolysis. This case recalls the potential importance of myocardial injury in the prognosis of malaria and prompts a reevaluation of current perspectives on the pathogenesis of severe falciparum infection. In the light of this, we have reviewed the cases of cardiac complications in malaria published to date.