Essential oil supplementation in milk replacers: short- and long-term impacts on feed efficiency, the faecal microbiota and the plasma metabolome in dairy calves.

Early supplementation with oregano essential oil (EO) in milk replacer (MR) may improve growth, immune responses, the microbiota and the metabolome in dairy calves during pre-weaning and in adulthood. Sixteen female dairy calves (3 days of age) were divided in two groups (n = 8/group): the control group (no EO) and the EO group (0.23 ml of EO in MR during 45 days). After weaning, calves were kept in a feedlot and fed ad libitum. The animals were weighed, and blood and faecal samples were collected on days 3 (T0), 45 (T1) and 370 (T2) to measure the biochemical profile and characterise peripheral blood mononuclear cells (PBMCs; CD4+, CD8+, CD14+, CD21+ and WC1+), the metabolome and microbiota composition. The EO group only had greater average daily weight gain during the suckling (EO supplementation) period (P = 0.030). The EO group showed higher average CD14+ population (monocytes) values, a lower abundance of Ruminococcaceae UCG-014, Faecalibacterium, Blautia and Alloprevotella and increased abundances of Allistipes and Akkermansia. The modification of some metabolites in plasma, such as butyric acid, 3-indole-propionic acid and succinic acid, particularly at T1, are consistent with intestinal microbiota changes. The data suggest that early EO supplementation increases feed efficiency only during the suckling period with notable changes in the microbiota and plasma metabolome; however, not all of these changes can be considered desirable from a gut health point of view. Additional research studies is required to demonstrate that EOs are a viable natural alternative to antibiotics for improving calf growth performance and health.

Multi-omics profiling of CSF from spinal muscular atrophy type 3 patients after nusinersen treatment: a 2-year follow-up multicenter retrospective study.

Spinal muscular atrophy (SMA) is a neurodegenerative disorder caused by mutations in the SMN1 gene resulting in reduced levels of the SMN protein. Nusinersen, the first antisense oligonucleotide (ASO) approved for SMA treatment, binds to the SMN2 gene, paralogue to SMN1, and mediates the translation of a functional SMN protein. Here, we used longitudinal high-resolution mass spectrometry (MS) to assess both global proteome and metabolome in cerebrospinal fluid (CSF) from ten SMA type 3 patients, with the aim of identifying novel readouts of pharmacodynamic/response to treatment and predictive markers of treatment response. Patients had a median age of 33.5 [29.5; 38.25] years, and 80% of them were ambulant at time of the enrolment, with a median HFMSE score of 37.5 [25.75; 50.75]. Untargeted CSF proteome and metabolome were measured using high-resolution MS (nLC-HRMS) on CSF samples obtained before treatment (T0) and after 2 years of follow-up (T22). A total of 26 proteins were found to be differentially expressed between T0 and T22 upon VSN normalization and LIMMA differential analysis, accounting for paired replica. Notably, key markers of the insulin-growth factor signaling pathway were upregulated after treatment together with selective modulation of key transcription regulators. Using CombiROC multimarker signature analysis, we suggest that detecting a reduction of SEMA6A and an increase of COL1A2 and GRIA4 might reflect therapeutic efficacy of nusinersen. Longitudinal metabolome profiling, analyzed with paired t-Test, showed a significant shift for some aminoacid utilization induced by treatment, whereas other metabolites were largely unchanged. Together, these data suggest perturbation upon nusinersen treatment still sustained after 22 months of follow-up and confirm the utility of CSF multi-omic profiling as pharmacodynamic biomarker for SMA type 3. Nonetheless, validation studies are needed to confirm this evidence in a larger sample size and to further dissect combined markers of response to treatment.

DDASSQ: An open-source, multiple peptide sequencing strategy for label free quantification based on an OpenMS pipeline in the KNIME analytics platform.

In this study we investigated the performance of a computational pipeline for protein identification and label free quantification (LFQ) of LC-MS/MS data sets from experimental animal tissue samples, as well as the impact of its specific peptide search combinatorial approach. The full pipeline workflow was composed of peptide search engine adapters based on different identification algorithms, in the frame of the open-source OpenMS software running within the KNIME analytics platform. Two different in silico tryptic digestion, database-search assisted approaches (X!Tandem and MS-GF+), de novo peptide sequencing based on Novor and consensus library search (SpectraST), were tested for the processing of LC-MS/MS raw data files obtained from proteomic LC-MS experiments done on proteolytic extracts from mouse ex vivo liver samples. The results from proteomic LFQ were compared to those based on the application of the two software tools MaxQuant and Proteome Discoverer for protein inference and label-free data analysis in shotgun proteomics. Data are available via ProteomeXchange with identifier PXD025097.

Alginate coating modifies the biological effects of cerium oxide nanoparticles to the freshwater bivalve Dreissena polymorpha.

The adsorption of biomacromolecules is a fundamental process that can alter the behaviour and adverse effects of nanoparticles (NPs) in natural systems. While the interaction of NPs with natural molecules present in the environment has been described, their biological impacts are largely unknown. Therefore, this study aims to provide a first evidence of the influence of biomolecules sorption on the toxicity of cerium oxide nanoparticles (CeO2NPs) towards the freshwater bivalve Dreissena polymorpha. To this aim, we compared naked CeO2NPs and coated with alginate and chitosan, two polysaccharides abundant in aquatic environments. Mussels were exposed to the three CeO2NPs (naked, chitosan- and alginate-coated) up to 14 days at 100 µg L-1, which is a concentration higher than the environmental one predicted for this type of NP. A suite of biomarkers related to oxidative stress and energy metabolism was applied, and metabolomics was also carried out to identify metabolic pathways potentially targeted by CeO2NPs. Results showed that the coating with chitosan reduced NP aggregation and increased the stability in water. Nonetheless, the Ce accumulation in mussels was similar in all treatments. As for biological effects, all three types of CeO2NPs reduced significantly the level of reactive oxygen species and the activity of superoxide dismutase, glutathione peroxidase and glutathione-S-transferase. The effect was more pronounced in individuals exposed to CeO2NPs coated with alginate, which also significantly induced the activity of the electron transport system. Metabolomics analysis of amino acid metabolism showed modulation only in mussels treated with CeO2NPs coated with alginate. In this group, 25 metabolites belonging to nucleotides, lipids/sterols and organic osmolytes were also modulated, suggesting that the nanoparticles affect energetic metabolism and osmoregulation of mussels. This study highlights the key role of the interaction between nanoparticles and natural molecules as a driver of nanoparticle ecotoxicity.

Determination of Cyanide by Microdiffusion Technique Coupled to Spectrophotometry and GC/NPD and Propofol by Fast GC/MS-TOF in a Case of Poisoning.

A man was found dead in a hotel located near Rome (Italy). The man was still holding a syringe attached to a butterfly needle inserted in his left forearm vein. The syringe contained a cloudy pinkish fluid. In the hotel room the Police found a broken propofol glass vial plus four sealed ones, an opened NaCl plastic vial and six more still sealed, and a number of packed smaller disposable syringes and needles. An opened plastic bottle containing a white crystalline powder labeled as potassium cyanide was also found. Systematic toxicological analysis (STA), carried out on blood, urine and bile, evidenced only the presence of propofol in blood and bile. So the validated L-L extraction protocol and the GC/MS-TOF method for the confirmation of propofol in the biological fluids optimized in our laboratory was applied to blood, urine and bile. The concentration of propofol resulted to be 0.432 µg/mL in blood and 0.786 µg/mL in bile. The quantitative determination of cyanide in blood was carried out by microdiffusion technique coupled to spectrophotometric detection obtaining a cyanide concentration of 5.3 µg/mL. The quantitative determination was then confirmed by GC/NPD and the concentration of cyanide resulted to be 5.5 µg/mL in blood and 1.7 µg/mL in bile. Data emerging from autopsy findings, histopathological exams and the concentrations of cyanide suggested that death might be due to poisoning caused by cyanide, however, respiratory depression caused by propofol could not be excluded.

Determination of Propofol by GC/MS and Fast GC/MS-TOF in Two Cases of Poisoning.

Two cases of suspected acute and lethal intoxication caused by propofol were delivered by the judicial authority to the Department of Sciences for Health Promotion and Mother-Child Care in Palermo, Sicily. In the first case a female nurse was found in a hotel room, where she lived with her mother; four 10 mg/mL vials and two 20 mg/mL vials of propofol were found near the decedent along with syringes and needles. In the second case a male nurse was found in the operating room of a hospital, along with a used syringe. In both cases a preliminary systematic and toxicological analysis indicated the presence of propofol in the blood and urine. As a result, a method for the quantitative determination of propofol in biological fluids was optimized and validated using a liquid-liquid extraction protocol followed by GC/MS and fast GC/MS-TOF. In the first case, the concentration of propofol in blood was determined to be 8.1 µg/mL while the concentration of propofol in the second case was calculated at 1.2 µg/mL. Additionally, the tissue distribution of propofol was determined for both cases. Brain and liver concentrations of propofol were, respectively, 31.1 and 52.2 µg/g in Case 1 and 4.7 and 49.1 µg/g in Case 2. Data emerging from the autopsy findings, histopathological exams as well as the toxicological results aided in establishing that the deaths were due to poisoning, however, the manner of death in each were different: homicide in Case 1 and suicide in Case 2.

A possible biomarker for methadone related deaths.

Methadone (MTH) concentrations in those dying of MTH toxicity totally overlap concentrations where the presence of MTH is only an incidental finding, making it very difficult to make distinctions in actual cases. A biomarker, be it anatomical or biochemical for MTH toxicity is badly needed, particularly if that markers were known to disrupt effective ventilation. Because the brainstem houses the regulatory centers for cardiorespiratory-control enters, it would seem to be the most likely anatomical site to seek abnormalities in cardiorespiratory control. OBJECTIVE: To locate and describe the cells of nucleus of the solitary tract (TS)(NTS) in human brainstem and determine if neuronal cell death, either necrotic or apoptotic, within the TS of humans is more common in deaths due directly to MTH toxicity than with in the solitary tract itself. DESIGN, SETTING, PARTICIPANTS: This was a single cohort study of MTH related decedents autopsied at a large university hospital. Each decedent had a recent history of non medical/illicit MTH use and had been pronounced dead in the field, prior to ever reaching the hospital. Complete autopsy and complete toxicology testing were performed on the formalin fixed brains of each individual. Multiple blocks were prepared of the area of interest, namely the tissue lying immediately between the inferior and the super colliculi. This volume, by definition, would have included the area of the Rostral Ventrolateral Medulla (RVLM), the location of the TS. Immunohistochemistry studies utilizing caspase-9 reaction (a protease enzyme involved in the process of preprogrammed death) were performed in order to estimate the degree and proportion of neuronal apoptosis, and also access the degree of classical necrosis within the NTS. MAIN OUTCOMES AND MEASURES: The primary outcome measure was the presence or absence of neuronal apoptosis and/or necrosis within the NTS. RESULTS: Cells displaying evidence of early apoptosis and advanced apoptosis, consisting primarily of nuclear fragmentation, admixed with other neurons displaying the features of classic necrosis were found. Evidence of classic necrosis was identifiable in most of the controls, though minor degrees of apoptosis were identifiable with Caspase staining and quantitative image analysis of immunohistochemical stains. CONCLUSIONS: and Relevance: Our study shows that neurons, primarily along the TS, but occasionally in other cell nuclei (even controls) are vulnerable, both to direct MTH toxicity (via apoptosis) and indirectly (via hypoxia leading to classical cell necrosis). When MTH is found to be present in significant concentrations, but apoptotic lesions are absent, it would be reasonable to assume that MTH was not primarily the cause of cardiorespiratory arrest.

DNA-based taxonomic identification of basidiospores in hallucinogenic mushrooms cultivated in "grow-kits" seized by the police: LC-UV quali-quantitative determination of psilocybin and psilocin.

The taxonomic identification of the biological material contained in the hallucinogenic mushrooms culture media, was carried out using a DNA-based approach, thus highlighting the usefulness of this approach in the forensic identification of illegal samples also when they are present as basidiospores mixed in culture media and spore-bearing fruiting body are not present. This approach is very useful as it allows the unequivocal identification of potentially illicit material before the cultivation and it enables to stop the material to the Customs and to destroy it due to its dangerousness without cultivating the "grow-kits" and without instructing a criminal case. In fact, even if psilocin and psilocybin and the whole mushrooms are illegal in many countries, there is no specific indication in the law about the so called "grow-kits", containing the spores. To confirm the data obtained by the taxonomic identification, a simple, reliable, efficient LC-UV method, using tryptamine as internal standard, suitable for the forensic quali-quantitative determination of psilocin and psilocybin in hallucinogenic mushroom was optimized, validated and applied to the mushrooms grown after the cultivation of the grow-kits seized by the judicial authority, with the authorization of the Ministry of Health. A cation exchange column was used in a gradient elution mode (Phase A: 50mMK2HPO4; 100mM NaCl pH=3 Phase B: methanol). The developed method was linear over the calibration range with a R(2)>0.9992 for both the analytes. The detection and quantification limits were respectively 0.01 and 0.1µg/mL for psilocybin and 0.05µg/mL and 0.1µg/mL for psilocin and the intra- and inter-day precision was satisfactory (coefficients of variation <2.0% for both the analytes). The content of psilocybin in the mushrooms grown from the seized "grow-kits" ranged from 1.02 to 7.60mg/g of dry vegetable material, while the content of psilocin from 0.415 to 8.36mg/g.

Unexpected variation of the codeine/morphine ratio following fatal heroin overdose.

Postmortem samples from 14 cases of suspected heroin overdose were subjected to a preliminary systematic toxicological analysis in order to highlight the presence of unknown exogenous compounds (e.g., drugs of abuse, alcohol) that may have played a role in the mechanism of death. This analysis unveiled histories of poly-drug use in seven of the cases under investigation. Moreover, the concentrations of morphine and codeine in the brain were also investigated, and the results were compared with the data obtained from the blood specimens. The concentration of morphine in blood ranged from 33 to 688 ng/mL, while the concentration of codeine ranged from 0 to 193 ng/mL. However, in the brain, the concentration of morphine was found to be between 85 and 396 ng/g, while the levels of codeine ranged from 11 to 160 ng/g. The codeine/morphine ratio in the blood ranged from 0.043 to 0.619; however, in the brain, the same ratio was found to be between 0.129 and 0.552. In most cases, a significantly higher codeine/morphine ratio was found in the brain, suggesting the accumulation of codeine in brain tissue due its high lipophilicity as compared with morphine.

Development and validation of a reliable method for studying the distribution pattern for opiates metabolites in brain.

Brain distribution pattern of "street" heroin metabolites (morphine and codeine) was investigated in two fatalities due to "acute narcotism". A suitable sample pretreatment prior to solid-phase-extraction was developed to achieve a good recovery of the analytes and to eliminate the interfering species. After derivatization with MSTFA, samples were analyzed by GC/MS. Specificity, accuracy, precision and linearity of the method were evaluated; LOD and LOQ were, respectively, 10ng/25ng for morphine and 5ng/10ng for codeine. This method was applied to the analysis of six brain areas (hippocampus, frontal lobe, occipital lobe, nuclei, bulb and pons) coming from two cases of heroin-related deaths. No evidence of accumulation of metabolites in a specific brain region was found.

Analysis of fluid extracts obtained from Papaver rhoeas petals contaminated with Papaver bracteatum petals.

In this paper, we report a case of misidentification of medicinal plants involving dried petals of Papaver rhoeas (red poppy) contaminated with Papaver bracteatum (scarlet poppy) petals. Preliminary TLC analysis indicated the presence of thebaine either in the fluid extracts or in the petals. It was therefore necessary to carry out an accurate botanic examination of the plant material, which revealed contamination of the red poppy petals with scarlet poppy petals. Moreover, to confirm the adulteration, we developed and validated an efficient, reversed-phase ion pair HPLC method for determination of the alkaloids specific for the Papaver species. Six petal batches and five commercial fluid extracts were analyzed. Only one petal batch from Iran contained thebaine and its analogue oripavine while the alkaloids typical for the Papaver bracteatum species were identified in all fluid extracts, meaning that they were all prepared with contaminated petals.

A case of fatal intoxication from metformin.

A case of fatal intoxication from metformin is presented. The decedent was an obese 58-year-old-woman with type II diabetes, in whom severe lactic acidosis secondary to metformin accumulation was precipitated by acute renal failure. She had been on metformin 500 mg twice a day. Postmortem blood was deproteinated with acetonitrile, washed with dichloromethane, and the resulting supernatant injected into high-performance liquid chromatography system. Separation was performed on a analytical 125 x 4 mm i.d. RP-8 column. The wavelength was set at 235 nm. The mobile phase was acetonitrile (40%), sodium lauryl sulfate, and sodium dihydrogen phosphate adjusted to pH 5.1 (60%) at a flow rate of 1.0 mL/min. The concentration of metformin in postmortem blood was 77.3 microg/mL. The qualitative result was also confirmed by LC/APCI/MS/MS analysis.

Validation of a GC/MS method for the determination of tramadol in human plasma after intravenous bolus.

Tramadol quantitative determination by gas chromatography-mass spectrometry (GC/MS) using nefopam hydrochloride as internal standard (IS) and two calibration curves because of the large range of concentration attended in the plasmatic samples is described. Plasma samples drawn from subjects in postoperative period treated with two different initial intravenous (iv) bolus of tramadol (50 and 100 mg) followed by tramadol at the same infusion rate (12 mg h(-1)) are analysed. We operated for the qualitative analysis in Scan mode while for the quantitative analysis in SIM mode, selecting the ion m/z 58 for tramadol and m/z 179 for IS. The limit of detection (LOD) was 0.01 microg ml(-1) and the limit of quantification was 0.04 microg ml(-1).