Search

Depression is currently the main disease which endangers human health and emotion. However, the existing antidepressants have the disadvantages of slowly taking effect and high recurrence rate. Numerous studies have shown that depression causes disorders in energy metabolism, but the specific metabolic pathways remain unclear. The stable isotope-resolved metabolomics (SIRM) can clarify the specific metabolic pathways of energy metabolism disorders in depression and provide a strong basis for the pathogenesis of depression and new targets for the development of new antidepressants. We applied this method to the chronic unpredictable mild stress (CUMS) model, and the metabolites related to energy metabolism were comprehensively analyzed on HILIC and T3 columns through LC-MS. Conventional untargeted metabolomics found 50 differential metabolites, and most of them were focused on the energy metabolism pathway. SIRM exhibited that 78 metabolites related to energy metabolism were labeled, and 28 of them were observed significantly changed in the model group compared with control. Our results revealed depression inhibited TCA cycle and activated gluconeogenesis pathway, and abnormally increased pyruvic acid may participate in pyrimidine biosynthesis, phospholipid synthesis, and amino acid metabolism pathways. Pyruvate carboxylase (PC), pyruvate dehydrogenase (PDH), aspartate aminotransferase (AST) and phosphoenolpyruvate carboxykinase (PEPC) may be the new targets for depression. We established a SIRM method at animal level. To the best of our knowledge, it is the first time to apply the method for the study of depression. The method is of great significance and value for further explaining the pathogenesis of depression and the development of antidepressants., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Effective and expansive methods for multiresidue pesticide analysis are desired for routine monitoring programs. These methods are complex, especially when several hundred pesticides are involved., Objective: Two approaches to sort data and identify isomers and isobaric ions in pesticide mixtures were evaluated to determine whether they could be differentiated by mass resolving power and/or chromatographic resolution., Method: This study presents an application of ultra-high performance liquid chromatography electrospray Q-Orbitrap mass spectrometry (UHPLC/ESI Q-Orbitrap) along with QuEChERS for the quantitation of 655 pesticide residues in fruits and vegetables., Results: From the developed method, 94.7% of the 655 pesticides in fruits and 93.9% of those in vegetables had recoveries between 81% and 110%; 98.3% in both fruits and vegetables had an intermediate precision of ≤20%; and 97.7% in fruits or 97.4% in vegetables showed measurement uncertainty of ≤50%. When the retention time difference (ΔtR) of two isomers was ≥0.12 min, they were chromatographically resolved. Twenty five out of 35 pairs or groups of isomers were chromatographically separated (ΔtR ≥ 0.12 min), but 14 pairs were not resolved (ΔtR < 0.12 min). There were 493 pairs of pesticides with a mass-to-charge difference of <1 Da. Only one pair of isobaric ions could not be separated by mass and chromatographic resolution., Highlights: UHPLC/ESI Q-Orbitrap along with QuEChERS sample preparation offers a practical quantitative companion method to a non-target data acquisition for target analysis workflow for pesticide residue analysis in routine monitoring programs for food safety., (© Her Majesty the Queen in Right of Canada, Canadian Food Inspection Agency, 2020.)

Rational design of pharmaceutical drugs targeting integral membrane G protein-coupled receptors (GPCR) requires thorough understanding of ligand binding and mechanism of activation through high resolution structural studies of purified proteins. Due to inherent conformational flexibility of GPCR, stabilization of these proteins solubilized from cell membranes into detergents is a challenging task. Here, we take advantage of naturally occurring post-translational modifications for stabilization of purified GPCR in detergent micelles. The recombinant cannabinoid CB 2 receptor was expressed at high yield in Expi293F mammalian cell cultures, solubilized and purified in Façade detergent. We report superior stability of the mammalian cell-expressed receptor compared to its E. coli-expressed counterpart, due to contributions from glycosylation of the N terminus and palmitoylation of the C terminus of CB 2 . Finally, we demonstrate that the mammalian Expi293F amino acid labelling kit is suitable for preparation of multi-milligram quantities of high quality, selectively stable isotope-labeled GPCR for studies by nuclear magnetic resonance.

Acylated compounds are often present in herbal medicines. In this study, a diagnostic product ion-based strategy was established to comprehensively characterize acylated compounds in Scrophulariae Radix. After untargeted data acquisition using ultra-high performance liquid chromatography coupled with Orbitrap mass spectrometry, the data were processed by three-stage diagnostic product ions. First, diagnostic product ions corresponding to the acyl groups (cinnamoyl, p-coumaroyl, feruloyl, and caffeoyl) were used to search 90 compounds. Second, these compounds were divided into three categories using diagnostic product ions for phenylethanoid glycosides, iridoid glycosides, and phenylpropanoids, respectively. Last, the linkage position of the acyl group to iridoid glycosides was discriminated via the third-stage diagnostic product ions. As a result, 90 acylated compounds were characterized, and 37 of them were reported from Scrophulariae Radix for the first time., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This work describes the holistic fingerprinting method based on liquid chromatography coupled with charged aerosol detection(CAD) to profile non-saponin from water-soluble parts and determination of dencichine in Panax ginseng(PG), P. quinquefolium(PQ) and P. notoginseng(PNG). Sample extraction was carried out by water with ultra sonication for 30 min, which was eluted by Retain PEP for further analysis. The analysis was performed on a Hypercarb of porous graphitized carbon(3.0 mm×150 mm, 3 μm) column with acetonitrile and 0.1% perfluoropentanoic acid as mobile phase at a flow rate of 0.8 mL·min~(-1). Temperature of evaporator and nitrogen pressure for CAD were set at 50 ℃and 60.1 psi(1 psi≈6.895 kPa), respectively. As a result, dencichine and other polar components had a good performance on resolution and retention. The correlation coefficient(R~2) of dencichine was 0.998 2 in the concentration from 0.019 2 to 0.48 μg·mL~(-1). Limit of quantitation calculated by signal to noise of 10 was 7.4 ng·mL~(-1), and the recovery ranged from 95.52% to 102.7%. Chemical profile of the water-soluble part from PG, PQ and PNG was similar holistically, while the relative content for dencichine and other partial components varied significantly. The proposed method was used for characteristic of chemical profiling for non-saponin from water-soluble part, and determination of dencichine in PG, PQ and PNG.

The UCSF Mouse Inventory Database Application is an open-source Web App that provides information about the mutant alleles, transgenes, and inbred strains maintained by investigators at the university and facilitates sharing of these resources within the university community. The Application is designed to promote collaboration, decrease the costs associated with obtaining genetically-modified mice, and increase access to mouse lines that are difficult to obtain. An inventory of the genetically-modified mice on campus and the investigators who maintain them is compiled from records of purchases from external sources, transfers from researchers within and outside the university, and from data provided by users. These data are verified and augmented with relevant information harvested from public databases, and stored in a succinct, searchable database secured on the university network. Here we describe this resource and provide information about how to implement and maintain such a mouse inventory database application at other institutions., (Copyright © 2020 Wall et al.)

Many phase transformations associated with solid-state precipitation look structurally simple, yet, inexplicably, take place with great difficulty. A classic case of difficult phase transformations is the nucleation of strengthening precipitates in high-strength lightweight aluminium alloys. Here, using a combination of atomic-scale imaging, simulations and classical nucleation theory calculations, we investigate the nucleation of the strengthening phase θ' onto a template structure in the aluminium-copper alloy system. We show that this transformation can be promoted in samples exhibiting at least one nanoscale dimension, with extremely high nucleation rates for the strengthening phase as well as for an unexpected phase. This template-directed solid-state nucleation pathway is enabled by the large influx of surface vacancies that results from heating a nanoscale solid. Template-directed nucleation is replicated in a bulk alloy as well as under electron irradiation, implying that this difficult transformation can be facilitated under the general condition of sustained excess vacancy concentrations.

To navigate within the geomagnetic field, magnetotactic bacteria synthesize magnetosomes, which are unique organelles consisting of membrane-enveloped magnetite nanocrystals. In magnetotactic spirilla, magnetosomes become actively organized into chains by the filament-forming actin-like MamK and the adaptor protein MamJ, thereby assembling a magnetic dipole much like a compass needle. However, in Magnetospirillum gryphiswaldense, discontinuous chains are still formed in the absence of MamK. Moreover, these fragmented chains persist in a straight conformation indicating undiscovered structural determinants able to accommodate a bar magnet-like magnetoreceptor in a helical bacterium. Here, we identify MamY, a membrane-bound protein that generates a sophisticated mechanical scaffold for magnetosomes. MamY localizes linearly along the positive inner cell curvature (the geodetic cell axis), probably by self-interaction and curvature sensing. In a mamY deletion mutant, magnetosome chains detach from the geodetic axis and fail to accommodate a straight conformation coinciding with reduced cellular magnetic orientation. Codeletion of mamKY completely abolishes chain formation, whereas on synthetic tethering of magnetosomes to MamY, the chain configuration is regained, emphasizing the structural properties of the protein. Our results suggest MamY is membrane-anchored mechanical scaffold that is essential to align the motility axis of magnetotactic spirilla with their magnetic moment vector and to perfectly reconcile magnetoreception with swimming direction.

Background: Gene mutation analysis from plasma circulating tumor DNA (ctDNA) can provide timely information regarding the mechanism of resistance that could translate to personalised treatment. We compared concordance rate of next generation sequencing (NGS) and droplet digital polymerase chain reaction (ddPCR) in the detection of the EGFR activating and T790M mutation from plasma ctDNA with diagnostic tissue biopsy-based assays. The second objective was to test whether putative osimertinib resistance associated mutations were detectable from plasma using NGS., Methods: From January 2016 to December 2017, we prospectively collected plasma samples from patients prior to commencement of second- or third-line osimertinib therapy and upon disease progression, in a single tertiary hospital in South Western Sydney, Australia. Amplicon-based NGS and ddPCR assays were used to detect activating epidermal growth factor receptor (EGFR) and T790M mutations in 18 plasma samples from nine patients; all patients were required to have tissue biopsies with known EGFR status., Results: High concordance of allelic fractions were seen in matched plasma NGS and ddPCR for activating EGFR mutations and T790M mutations (R 2 = 0.92, P < 0.0001). Using tissue biopsies as reference standard, sensitivity was 100% for NGS and 94% for ddPCR. Several possible osimertinib resistance associated mutations, including PIK3CA, BRAF and TP53 mutations, were detected by NGS in samples upon progression on osimertinib therapy., Conclusion: ddPCR assays for EGFR mutations appear to be as sensitive and highly concordant as amplicon-based NGS. NGS has the ability to detect novel resistance mutations., (© 2019 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.)

The emergence of Massively Parallel Sequencing technologies enabled the analysis of full mitochondrial (mt)DNA sequences from forensically relevant samples that have, so far, only been typed in the control region or its hypervariable segments. In this study, we evaluated the performance of a commercially available multiplex-PCR-based assay, the Precision ID mtDNA Whole Genome Panel (Thermo Fisher Scientific), for the amplification and sequencing of the entire mitochondrial genome (mitogenome) from even degraded forensic specimens. For this purpose, more than 500 samples from 24 different populations were selected to cover the vast majority of established superhaplogroups. These are known to harbor different signature sequence motifs corresponding to their phylogenetic background that could have an effect on primer binding and, thus, could limit a broad application of this molecular genetic tool. The selected samples derived from various forensically relevant tissue sources and were DNA extracted using different methods. We evaluated sequence concordance and heteroplasmy detection and compared the findings to conventional Sanger sequencing as well as an orthogonal MPS platform. We discuss advantages and limitations of this approach with respect to forensic genetic workflow and analytical requirements., (Copyright © 2019. Published by Elsevier B.V.)

Background: Approximately two thirds of patients with localized triple-negative breast cancer (TNBC) harbor residual disease (RD) after neoadjuvant chemotherapy (NAC) and have a high risk-of-recurrence. Targeted therapeutic development for TNBC is of primary significance as no targeted therapies are clinically indicated for this aggressive subset. In view of this, we conducted a comprehensive molecular analysis and correlated molecular features of chemorefractory RD tumors with recurrence for the purpose of guiding downstream therapeutic development., Methods: We assembled DNA and RNA sequencing data from RD tumors as well as pre-operative biopsies, lymphocytic infiltrate, and survival data as part of a molecular correlative to a phase II post-neoadjuvant clinical trial. Matched somatic mutation, gene expression, and lymphocytic infiltrate were assessed before and after chemotherapy to understand how tumors evolve during chemotherapy. Kaplan-Meier survival analyses were conducted categorizing cancers with TP53 mutations by the degree of loss as well as by the copy number of a locus of 18q corresponding to the SMAD2, SMAD4, and SMAD7 genes., Results: Analysis of matched somatic genomes pre-/post-NAC revealed chaotic acquisition of copy gains and losses including amplification of prominent oncogenes. In contrast, significant gains in deleterious point mutations and insertion/deletions were not observed. No trends between clonal evolution and recurrence were identified. Gene expression data from paired biopsies revealed enrichment of actionable regulators of stem cell-like behavior and depletion of immune signaling, which was corroborated by total lymphocytic infiltrate, but was not associated with recurrence. Novel characterization of TP53 mutation revealed prognostically relevant subgroups, which were linked to MYC-driven transcriptional amplification. Finally, somatic gains in 18q were associated with poor prognosis, likely driven by putative upregulation of TGFß signaling through the signal transducer SMAD2., Conclusions: We conclude TNBCs are dynamic during chemotherapy, demonstrating complex plasticity in subclonal diversity, stem-like qualities, and immune depletion, but somatic alterations of TP53/MYC and TGFß signaling in RD samples are prominent drivers of recurrence, representing high-yield targets for additional interrogation.

Dust is a fate of many contaminants and may be an important medium for the human exposure to these contaminants. Microplastics (MPs) have been observed in dust in previous studies. However, the mass concentrations of dominant MPs in dust and the exposure risk to human remain unclear. In this study, indoor and outdoor dust samples were collected from 39 major cities of China. The mass concentrations of polyethylene terephthalate (PET) and polycarbonate (PC) MPs were determined through alkali-assisted thermal depolymerization-liquid chromatography-tandem mass spectrometry, and the shape and component distribution of MPs were analyzed by optical microscopy and micro-Fourier transform infrared spectroscopy. PET MPs were detected in all the samples at high concentrations of 1550-120,000 mg/kg (indoor) and 212-9020 mg/kg (outdoor) and PC MPs were detected in approximately 70% of the samples, with median concentrations of 4.6 mg/kg (indoor) and 2.0 mg/kg (outdoor). Fiber was the main shape of suspected MPs, and polyester (including PET) was identified as an important component in MPs from dust. Indoor dust is a non-negligible source of human exposure to MPs, accounting for a geomean daily intake of 17,300 ng/kg-bw of PET MPs in children., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Twinning structures and their interfacial segregation play a key role in strengthening of magnesium alloys. Micro-steps are frequently existed in the incoherent twin boundaries, while the effect of them on interface and interfacial segregation is still not clear. In this work, we performed an atomic-scale microstructure analysis using high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) to explore the effect of micro-steps on twin and its interfacial segregation in Mg-Ag alloy. Diffraction pattern of the incoherent {10 1 ¯ 1} twin shows that the misorientation has a slight tilt of 5° from its theoretical angle of 125° due to the accumulated effects of the micro-steps and their misfit dislocations in twin boundaries. Most of the micro-steps in {10 1 ¯ 1} twin boundary are in the height of 2 d ( 10 1 ¯ 1 ) and 4 d ( 10 1 ¯ 1 ) , respectively, and both of them have two types according to whether there are dislocations on the micro-steps. The twin boundary is interrupted by many micro-steps, which leads to a step-line distributed interfacial segregation. Moreover, the Ag tends to segregate to dislocation cores, which results in the interruption of interfacial segregation at the micro-steps with dislocations.

Mutation in leucine-rich repeat kinase 2 (LRRK2) is a common cause of familial Parkinson's disease (PD). Recently, we showed that a disease-associated mutation R1441H rendered the GTPase domain of LRRK2 catalytically less active and thereby trapping it in a more persistently "on" conformation. However, the mechanism involved and characteristics of this on conformation remained unknown. Here, we report that the Ras of complex protein (ROC) domain of LRRK2 exists in a dynamic dimer-monomer equilibrium that is oppositely driven by GDP and GTP binding. We also observed that the PD-associated mutations at residue 1441 impair this dynamic and shift the conformation of ROC to a GTP-bound-like monomeric conformation. Moreover, we show that residue Arg-1441 is critical for regulating the conformational dynamics of ROC. In summary, our results reveal that the PD-associated substitutions at Arg-1441 of LRRK2 alter monomer-dimer dynamics and thereby trap its GTPase domain in an activated state.

We evaluated whether the excluded stomach (ES) after Roux-en-Y gastric bypass (RYGB) can represent a premalignant environment. Twenty obese women were prospectively submitted to double-balloon enteroscopy (DBE) with gastric juice and biopsy collection, before and 3 months after RYGB. We then evaluated morphological and molecular changes by combining endoscopic and histopathological analyses with an integrated untargeted metabolomics and transcriptomics multiplatform. Preoperatively, 16 women already presented with gastric histopathological alterations and an increased pH (≥4.0). These gastric abnormalities worsened after RYGB. A 90-fold increase in the concentration of bile acids was found in ES fluid, which also contained other metabolites commonly found in the intestinal environment, urine, and faeces. In addition, 135 genes were differentially expressed in ES tissue. Combined analysis of metabolic and gene expression data suggested that RYGB promoted activation of biological processes involved in local inflammation, bacteria overgrowth, and cell proliferation sustained by genes involved in carcinogenesis. Accumulated fluid in the ES appears to behave as a potential premalignant environment due to worsening inflammation and changing gene expression patterns that are favorable to the development of cancer. Considering that ES may remain for the rest of the patient's life, long-term ES monitoring is therefore recommended for patients undergoing RYGB.

Background: Sensitization to allergen components has been linked to asthma in children, but studies in adults are lacking., Objective: To study the relation of sensitization to furry animal allergen components to risk of asthma, rhinitis, and markers of asthma severity in adults., Methods: From the West Sweden Asthma Study, a random population-representative sample of adults aged 16 to 75 years, 2006 participants were clinically examined; 1872 were analyzed for serum IgE level to a mix of aeroallergens. Those with an IgE level of more than 0.35 kU A /L to cat, dog, or horse allergen components were analyzed for specific cat (Felis domesticus [Fel d 1, Fel d 2, and Fel d 4]), dog (Canis familiaris [Can f 1, Can f 2, Can f 3, and Can f 5]), and horse (Equus caballus [Equ c 1]) allergen components. We defined monosensitization, double sensitization, and polysensitization (>2 components) patterns and applied cluster analysis to derive distinct sensitization clusters., Results: Sensitization to each allergen component, lipocalins, each sensitization pattern, and each sensitization cluster (nonsensitized, Fel d 1-driven sensitized, and multisensitized clusters) was associated with substantial increased risk of asthma, rhinitis, concomitant asthma and rhinitis, and Asthma Control Test-controlled asthma. Fel d 1, Can f 1, Can f 2, Can f 3, polysensitization, and multisensitized cluster were further associated with increased fractional exhaled nitric oxide and eosinophil levels, but with lower PD 20 methacoline (provocative dose of methacholine causing a 20% drop in FEV 1 ) values. There was no association with asthma exacerbations, FEV 1 predicted values, emergency visits or regular oral steroid use, and neutrophil levels., Conclusions: Sensitization to furry animal allergen components is an important predictor of asthma, rhinitis, and markers of asthma severity with increased blood eosinophils, fractional exhaled nitric oxide, and airway hyperreactivity., (Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

A non-target data acquisition for target analysis (nDATA) workflow based on accurate mass measurements using UHPLC/ESI Q-Orbitrap full MS-data-independent acquisition and a compound database was developed to screen pesticide residues in fruit and vegetable samples. The compound database of 845 pesticides was built from dd-MS 2 (data-dependent acquisition) product ion spectral data and LC retention times of individual pesticide standards. MS 2 spectra of samples were acquired using multiplexing data-independent acquisition (mDIA) and variable data-independent acquisition (vDIA). Screening of pesticides in samples was based on either the retention time (± 0.5 min) and the mass accuracy (± 5 ppm) of a precursor (RTP by full MS) or the retention time (± 0.5 min) and the mass accuracy (± 5 ppm) of a precursor and its fragment ion (RTFI by full MS/DIA). In validation studies involving mDIA and vDIA analysis of 10 fruits and vegetables spiked with pesticides prior to QuEChERS sample preparation, RTP correctly found up to 765 and 796 pesticides at 10 and 100 μg/kg, respectively, whereas RTFI correctly identified up to 729 and 764 pesticides at the same respective concentrations. UHPLC/ESI Q-Orbitrap full MS/mDIA or vDIA proved to be a comprehensive detection technique and has potential for pesticide residue screening in fruits and vegetables. Graphical Abstract.

Electrochemical reduction is an eco-friendly alternative for energy-saving artificial N 2 fixation. The development of this process requires efficient N 2 reduction reaction (NRR) electrocatalysts to overcome the challenge with N 2 activation. We show that a Cr 2 O 3 nanoparticle-reduced graphene oxide hybrid (Cr 2 O 3 -rGO) is as an outstanding catalyst for electrochemical N 2 -to-NH 3 conversion under ambient conditions. In 0.1 M HCl, Cr 2 O 3 -rGO achieves a high NH 3 yield of 33.3 μg h -1 mg -1 cat. at -0.7 V vs RHE and a high Faradaic efficiency of 7.33% at -0.6 V vs RHE, with excellent selectivity for NH 3 synthesis and stability. Density functional theory calculations were executed to gain further insight into the mechanisms.

There is an ongoing urgent need for new targeted antibacterial compounds with novel mechanisms of action for the treatment of infections caused by bacteria that are resistant to currently available materials. Since the expression of glycosidase enzymes within bacteria is unequally distributed, glycoside derivatives of antibacterial agents offer potential as targeted prodrugs for bacterial infections. Herein we report the synthesis and characterisation of four α-D-glycopyranosides and three β-D-glycopyranosides of the broad antibacterial agent triclosan, in generally good synthetic yields, and with excellent purities. Each glycoside was analysed to determine its ability to inhibit the growth of a wide range of Gram-negative and Gram-positive organisms, including many of clinical significance. All of the triclosan glycosides that were synthesized demonstrated antibacterial activity against many of the organisms that were examined. For example, β-galactoside (3a) and α-arabinoside (3c) had MIC values of 0.5 μg/ml for several strains of S. aureus and S. haemolyticus. The triclosan glycosides were also generally found to be more water soluble and much more selective than the underivatized triclosan, making them ideal both for the targeted inhibition of bacterial growth and as agents for the selective recovery of bacteria from mixed cultures. In the latter case, two Bacillus strains could be identified from various strains of Bacillus and Staphylococcus after inoculation onto Nutrient Agar No. 2 with 0.25 μg/ml triclosan-α-D-glucopyranoside (3e). This glucoside may, therefore, be of use for the isolation and identification of the food-poisoning organism Bacillus cereus., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Background: Clinical Breast Examination (CBE) is the examination of a women's breasts by a healthcare professional, such as a breast surgeon, family physician or breast-care nurse who is trained to recognise many different types of abnormalities and warning signs in the breast [1]. CBE is particularly important in rural areas and developing countries who have limited access to technology such as mammography. CBE needs to be taught to health professionals like any other clinical skill used by medical professionals in the workplace. CBE in part involves palpation of the breast, that is, determining by touch which breast lumps are normal and which are suspicious in feeling. The gold standard for assessing tactile skills in CBE is seeing whether students can accurately identify and discriminate between different breast lumps also known as masses (IDBM) on actual patients in a clinical setting. However, this is not practical in a medical education setting. Usually the testing methods 'go through the motions' of feeling the breast as part of CBE. So the students' technique is examined either using unrealistic simulation models or using an intimate examination associate (IEA), an actor/volunteer who permits students to examine their intimate body parts such as breast or genitals for teaching purposes. These volunteers do not have any abnormalities so this teaching does not include the actual detection of suspicious lumps. We undertook a study of clinical skill with 10 medical students to examine different methods of assessing novice student clinical skills after a brief training in CBE., Objectives: This study aims to evaluate the effectiveness of current training and assessment of novice students in CBE and their capacity to identify and discriminate breast masses (IDBM) on actual patients., Methods: We assessed each student's IDBM ability in an actual clinical situation, a breast clinic with a mixture of eight IEAs and one real patient with a large, easily palpable, putative breast cancer. We recruited 10 clinically inexperienced medical students, who were trained for 30 minutes by two breast surgeons using an IEA. Students were tested in a simulated clinical setting, a breast clinic where each examined 4 IEAs and one patient. The students were blind to who was the real patient and who was an IEA. Patients were examined by a breast surgeon in private prior to the commencement in the study. The breast surgeon recorded any clinical finding on the patients during the initial examination. The surgeon coached each patient on how to mark the students and showed the patient their results so the patients had a benchmark. After each examination was finished the students had four different assessments: 1) patients marked each student, 2) students were independently proctored - that is, marked by an expert, 3) students recorded their clinical findings and 4) students recorded how confident they were that they had the correct findings. Results from different kinds of student assessments were compared.RESULTSA chi-square test for independence between true positive or negative masses versus student-assessed positive or negative masses was not significant at alpha = .05. This means that there was no statistical association in the indication of positive or negative presence of masses versus whether such masses were actually present or absent. By comparison, experts (breast surgeons) were able to detect normal and abnormal breast masses by palpation alone 100% of the time and rate their confidence level as 'certain'. Unlike the experts, student self-reported confidence was unrelated to their competence score (CS). Proctoring was inversely related to the students' CS.CONCLUSIONSThe main conclusion is that novice students do not seem to be able to accurately detect breast masses in a clinical setting even after training. On the basis of these results, we believe that a comprehension component in the current CBE testing is needed in addition to the current methods of testing.

Isocitrate dehydrogenase 1 (IDH1) mutations in gliomas, fibrosarcoma, and other cancers leads to a novel metabolite, D-2-hydroxyglutarate, which is proposed to cause tumorigenesis. The production of this metabolite also causes vulnerabilities in cellular metabolism, such as lowering NADPH levels. To exploit this vulnerability, we treated glioma and fibrosarcoma cells that harbor an IDH1 mutation with an inhibitor of nicotinamide adenine dinucleotide (NAD⁺) salvage pathway, FK866, and observed decreased viability in these cells. To understand the mechanism of action by which the inhibitor FK866 works, we used Raman imaging microscopy and identified that proteins and lipids are decreased upon treatment with the drug. Raman imaging showed a different distribution of lipids throughout the cell in the presence of the drug compared with the untreated cells. We employed nuclear magnetic resonance NMR spectroscopy and mass spectrometry to identify the classes of lipids altered. Our combined analyses point to a decrease in cell division due to loss of lipid content that contributes to membrane formation in the in vitro setting. However, the FK866 drug did not have the same potency in vivo. The use of Raman imaging microscopy indicated an opposite trend of lipid distribution in the tissue collected from treated versus untreated mice when compared with the cells. These results demonstrate the role of Raman imaging microscopy to identify and quantify metabolic changes in cancer cells and tissue.

Subsequent to conducting the Chromosome-Centric Human Proteome Project, we have focused on human testis-enriched missing proteins (MPs) since 2015. For protein coverage to be enhanced, a multiprotease strategy was used for separation of samples by 10% SDS-PAGE. For the separating efficiency to be improved, a high-pH reverse phase (RP) separation strategy was applied to fractionate complex samples in this study. A total of 11,558 proteins was identified, which is the largest proteome data set for single human tissue sample so far. On the basis of this large-scale data set, we verified 14 MPs (PE2) in neXtProt (2018-01) after spectrum quality analysis, isobaric post-translational modification, and single amino acid variant filtering, and synthesized peptide matching. Tissue expression analysis showed that 3 of 14 MPs were testis-specific proteins. Functional analysis showed that 10 of 14 MPs were closely related to liver tumor, liver carcinoma, and hepatocellular carcinoma. Another 100 MPs were listed as candidates but required additional verification information. All MS data sets have been deposited into the ProteomeXchange with the identifier PXD009737.

Background: Integrated molecular profiling has identified intrinsic expression-based bladder cancer molecular subtypes. Despite frequent histological diversity, robustness of subtypes in paired conventional (urothelial) and squamous components of the same bladder tumor has not been reported., Objective: To assess the impact of histological heterogeneity on expression-based bladder cancer subtypes., Design, Setting, and Participants: We performed clinically applicable, targeted DNA and/or RNA sequencing (multiplexed DNA and RNA sequencing [mxDNAseq and mxRNAseq, respectively]) on 112 formalin-fixed paraffin-embedded (FFPE) bladder cancer samples, including 12 cases with paired urothelial/squamous components and 21 bladder cancer cell lines., Outcome Measurements and Statistical Analysis: Unsupervised hierarchical and consensus clustering of target gene expression enabled derivation of basal/luminal molecular subtyping., Results and Limitation: Across 21 bladder cancer cell lines, our custom mxRNAseq panel was highly concordant with whole transcriptome sequencing, and assessed targets robustly determined expression-based basal/luminal subtypes from The Cancer Genome Atlas data (in silico) and internally sequenced FFPE tissues. Frequent deleterious TP53 (56%) and activating hotspot PIK3CA (30%) somatic mutations were seen across 69 high-quality tissue samples. Potentially targetable focal ERBB2 (6%) or EGFR (6%) amplifications were also identified, and a novel subgene copy-number detection approach is described. Combined DNA/RNA analysis showed that focally amplified samples exhibit outlier EGFR and ERBB2 expression distinct from subtype-intrinsic profiles. Critically, paired urothelial and squamous components showed divergent basal/luminal status in three of 12 cases (25%), despite identical putatively clonal prioritized somatic genomic alterations. Limitations include lack of profiled paired normal tissues for formal somatic alteration determination, and the need for formal analytical and clinical validation., Conclusions: Our results support the feasibility of clinically relevant integrative bladder cancer profiling and challenge the intrinsic nature of expression subtypes in histologically diverse bladder cancers., Patient Summary: A targeted RNA sequencing assay is capable of assessing gene expression-based subtypes in individual components of clinical bladder cancer tissue specimens. Different histological components of the same tumor may yield divergent expression profiles, suggesting that expression-based subtypes should be interpreted with caution in heterogeneous cancers., (Copyright © 2018 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

Hops and the components extracted from them are well known antibacterial agents used in beers and as food preservatives, in formulations for topical applications on their own or together with other antimicrobial agents, in hormone replacement therapy, as antioxidants, tumor development antagonists, and angiogenesis inhibitors. Their shortcomings: very low bioavailability, bitter taste, and susceptibility to oxidative decomposition have limited their applications. We propose nanosized chitosan, an inexpensive, readily available biopolymer with a broad spectrum of antibacterial activity, as carrier for lupulone (L) and xanthohumol (X), two components of hops. Chitosan nanoparticles (CNP) and chitosan-based nanocomposites encapsulating lupulone (CNL) and xanthohumol (CNX) were prepared by ionotropic gelation using sodium tripolyphosphate (TPP) as crosslinker. Different preparative ratios and conditions were investigated and the nanoparticles obtained were characterized by FTIR, colloidal titration, size, zeta potential, and antimicrobial activity. The kinetics of the release of L/X from composites was studied in vitro. All the nanoparticles were active against several Gram-positive, Gram-negative, and Candida strains. Synergistic interactions were observed in all cases, although hops are known mainly for their activity against Gram-positive bacteria. All nanoparticles showed good stability over several months., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: The widespread global access to antiretroviral drugs has led to considerable reductions in morbidity and mortality but, unfortunately, the risk of virologic failure increases with the emergence, and potential transmission, of drug resistant viruses. Detecting and quantifying HIV-1 drug resistance has therefore become the standard of care when designing new antiretroviral regimens. The sensitivity of Sanger sequencing-based HIV-1 genotypic assays is limited by its inability to identify minority members of the quasispecies, i.e., it only detects variants present above ~ 20% of the viral population, thus, failing to detect minority variants below this threshold. It is clear that deep sequencing-based HIV-1 genotyping assays are an important step change towards accurately monitoring HIV-infected individuals., Methods: We implemented and verified a clinically validated HIV-1 genotyping assay based on deep sequencing (DEEPGEN™) in two clinical laboratories in the United Kingdom: St. George's University Hospitals Healthcare NHS Foundation Trust (London) and at NHS Lothian (Edinburgh), to characterize minority HIV-1 variants in 109 plasma samples from ART-naïve or -experienced individuals., Results: Although subtype B HIV-1 strains were highly prevalent (44%, 48/109), most individuals were infected with non-B subtype viruses (i.e., A1, A2, C, D, F1, G, CRF02&#95;AG, and CRF01&#95;AE). DEEPGEN™ was able to accurately detect drug resistance-associated mutations not identified using standard Sanger sequencing-based tests, which correlated significantly with patient's antiretroviral treatment histories. A higher proportion of minority PI-, NRTI-, and NNRTI-resistance mutations was detected in NHS Lothian patients compared to individuals from St. George's, mainly M46I/L and I50 V (associated with PIs), D67 N, K65R, L74I, M184 V/I, and K219Q (NRTIs), and L100I (NNRTIs). Interestingly, we observed an inverse correlation between intra-patient HIV-1 diversity and CD4 + T cell counts in the NHS Lothian patients., Conclusions: This is the first study evaluating the transition, training, and implementation of DEEPGEN™ between three clinical laboratories in two different countries. More importantly, we were able to characterize the HIV-1 drug resistance profile (including minority variants), coreceptor tropism, subtyping, and intra-patient viral diversity in patients from the United Kingdom, providing a rigorous foundation for basing clinical decisions on highly sensitive and cost-effective deep sequencing-based HIV-1 genotyping assays in the country.

Chronic hyperlipidemia causes the dysfunction of pancreatic β-cells, such as apoptosis and impaired insulin secretion, which are aggravated in the presence of hyperglycemia. The underlying mechanisms, such as endoplasmic reticulum (ER) stress, oxidative stress and metabolic disorders, have been reported before; however, the time sequence of these molecular events is not fully understood. Here, using isobaric labeling-based mass spectrometry, we investigated the dynamic proteomes of INS-1 cells exposed to high palmitate in the absence and presence of high glucose. Using bioinformatics analysis of differentially expressed proteins, including the time-course expression pattern, protein-protein interaction, gene set enrichment and KEGG pathway analysis, we analyzed the dynamic features of previously reported and newly identified lipotoxicity- and glucolipotoxicity-related molecular events in more detail. Our temporal data highlight cholesterol metabolism occurring at 4 h, earlier than fatty acid metabolism that started at 8 h and likely acting as an early toxic event highly associated with ER stress induced by palmitate. Interestingly, we found that the proliferation of INS-1 cells was significantly increased at 48 h by combined treatment of palmitate and glucose. Moreover, benefit from the time-course quantitative data, we identified and validated two new molecular targets: Setd8 for cell replication and Rhob for apoptosis, demonstrating that our temporal dataset serves as a valuable resource to identify potential candidates for mechanistic studies of lipotoxicity and glucolipotoxicity in pancreatic β-cells., (© 2018 Li et al.)

Massively parallel sequencing is transforming forensic work by allowing various useful forensic markers, such as STRPs and SNPs, to be multiplexed providing information on ancestry, individual and familial identification, phenotypes for eye/hair/skin pigmentation, and the deconvolution of mixtures. Microhaplotypes also become feasible with massively parallel sequencing, these are DNA segments (smaller than 300 nucleotides) that are selected to contain multiple SNPs unambiguously defining three or more haplotype alleles occurring at common frequencies. The physical extent of a microhaplotype can thus be covered by a single sequence read making these loci phase-known codominant genetic systems. Such microhaplotypes supply significantly more information than a single SNP can. Our efforts to develop useful sets of microhaplotypes have already identified 182 such loci that we have studied on a large number of human populations from around the world. We present various analyses on 83 populations in our ongoing study for a subset of the best microhaplotypes currently available illustrating their characteristics and potential utility for ancestry, identification, and mixture deconvolution., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Since 1986, use of a Bovine International Standard (BIS) for bovine tuberculin has been required to ensure national and international uniformity regarding the potency designation of bovine tuberculin Purified Protein Derivative (PPDb) preparations produced by multiple manufacturers. The BIS is the unique golden standard in the guinea pig potency assay, representing 100% potency, where potencies of production batches are calculated as relative potencies in comparison with the potency of the BIS which was set at 32,500 international Unit (IU) per mg. The stock supply and lifetime of the BIS is limited.The aim of this study was to develop a model to determine the potency of a newly produced in-house Reference Standard (RS) for PPDb with great accuracy in the target species (cattle) and to prove its precision and accuracy in the guinea pig potency test. First simulations were done to estimate the required number of cattle needed. Then, 30 naturally bTB infected cattle were subjected to a tuberculin skin test using multiple injections of both the RS and the BIS. Both were applied randomly in the same volume and concentration (1 dose). The potency of the RS against the BIS was directly derived from the least square means (LSMEANS) and was estimated as 1.067 (95% CI: 1.025-1.109), equal to a potency of 34,700 ± 1,400 IU/mg. In six guinea pig potency assays the RS was used to assign potencies to production batches of PPDb. Here, precision and accuracy of the RS was determined according to the parallel-line assay. Relative potencies were estimated by exponentiation of the common slope. The corresponding 95% confidence intervals were obtained according to Fieller's theorem. In sensitized guinea pigs, the relative potency of the RS against the BIS was 1.115 (95% CI: 0.871-1.432), corresponding to an absolute potency of 36,238 IU/mg (95% CI: 28,308-46,540).In conclusion: the method used to determine the potency of the RS against the BIS in naturally bTB infected cattle, resulted in a highly accurate potency estimate of the RS. The RS can be used in the guinea pig test to assign potencies to PPDb production batches with high precision and accuracy.

Source apportionment studies of particulate matter (PM) link chemical composition to emission sources, while health risk analyses link health outcomes and chemical composition. There are limited studies to link emission sources and health risks from ambient measurements. We show such an attempt for particulate trace elements. Elements in PM 2.5 were measured in wintertime Beijing, and the total concentrations of 14 trace elements were 1.3-7.3 times higher during severe pollution days than during low pollution days. Fe, Zn, and Pb were the most abundant elements independent of the PM pollution levels. Chemical fractionation shows that Pb, Mn, Cd, As, Sr, Co, V, Cu, and Ni were present mainly in the bioavailable fraction. Positive matrix factorization was used to resolve the sources of particulate trace elements into dust, oil combustion, coal combustion, and traffic-related emissions. Traffic-related emission contributed 65% of total mass of the measured elements during low pollution days. However, coal combustion dominated (58%) during severe pollution days. By combining element-specific health risk analyses and source apportionment results, we conclude that traffic-related emission dominates the health risks by particulate trace elements during low pollution days, while coal combustion becomes equally or even more important during moderate and severe pollution days.

As a major group of plasticizers and flame-retardants, organophosphate esters (OPEs) have attracted particular attention due to their wide occurrence and potential impacts on human health and ecosystems. In the present study, the occurrence and distribution of 14 OPEs, including seven Alkyl-OPEs, three Cl-OPEs, and four Aryl-OPEs, were investigated in 65 road dust samples collected from November to December 2014 in Beijing, China. Cl-OPEs were the predominant compounds in the road dust samples, with the median concentration of 646 μg/kg, followed by the Alkyl-OPEs (median 135 μg/kg) and Aryl-OPEs (median 129 μg/kg). Tris(2-chloro-1-methylethyl) phosphate (TCPP) was the most abundant OPE with the median concentration of 384 μg/kg. In addition, OPEs levels showed significant difference (p < 0.05) in the spatial distribution. Markedly higher levels of OPEs were observed in 2nd and 3rd ring road with heavy traffic and high population density, indicating that the traffic and population were important factors for this distribution pattern. It was further supported by the analysis of OPEs in roadside soil and indoor dust in the vicinity of road dust sample sites. Finally, the average daily dose (ADD) for OPEs via inhalation, dermal absorption, and ingestion was calculated to evaluate the carcinogenic and non-carcinogenic risks to residents exposed to OPEs in the road dust. Risk assessment revealed that the risk originating from exposure to OPEs of road dust is currently low in Beijing, China., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

This paper presents a multi-class target screening method for the detection of 105 veterinary drug residues from 11 classes in milk using ultra-high performance liquid chromatography electrospray ionization quadrupole Orbitrap mass spectrometry (UHPLC/ESI Q-Orbitrap). The method is based on a non-target approach of full mass scan and multiplexing data-independent acquisition (Full MS/mDIA). The veterinary drugs include endectocides, fluoroquinolones, ionophores, macrolides, nitroimidazole, NSAIDs, β-lactams, penicillins, phenicols, sulfonamides, and tetracyclines. Veterinary drug residues were extracted from milk using a salting-out and solid-phase extraction (SOSPE) procedure, which entailed the precipitation of milk proteins by an extraction buffer (oxalic acid and EDTA, pH 3) and acetonitrile, a salting-out acetonitrile/water phase separation using ammonium sulfate, and solid-phase extraction for clean-up using polymeric reversed-phase sorbent cartridges. The Q-Orbitrap Full MS/dd-MS 2 (data-dependent acquisition) was used to acquire product-ion spectra of individual veterinary drugs to build a compound database and a mass spectral library, whereas its Full MS/mDIA was utilized to acquire sample data from milk for target screening of veterinary drugs fortified at 1.0 or 10.0 μg/kg. The in-spectrum mass correction or solvent background lock-mass correction was used to minimize mass error when building the compound database from experimental dd-MS 2 accurate mass data. Retention time alignment and response threshold adjustment were used to eliminate or reduce false negatives and/or false positive rates. The validated method was capable of screening 58% and 96% of 105 veterinary drugs at 1.0 and 10.0 μg/kg, respectively, without manually evaluating every compound during data processing, which will reduce the workload in routine practice.

Affinity mass spectrometry using selective proteolytic excision and extraction combined with MALDI and ESI mass spectrometry has been applied to the identification of epitope binding sites of lactose, GalNac, and blood group oligosaccharides in two blood group-specific lectins, human galectin-3 and glycine max lectin. The epitope peptides identified comprise all essential amino acids involved in carbohydrate recognition, in complete agreement with available X-ray structures. Tryptic and chymotryptic digestion of lectins for proteolytic extraction/excision-MS was substantially improved by pressure-enhanced digestion using an automated Barocycler procedure (40 kpsi). Both previously established immobilization on affinity microcolumns using divinyl sulfone and coupling of a specific peptide glycoprobe to the gold surface of a biosensor chip were successfully employed for proteolytic excision and extraction of carbohydrate epitopes and affinity measurements. The identified epitope peptides could be differentiated according to the carbohydrate employed, thus demonstrating the specificity of the mass spectrometric approach. The specificities of the epitope ligands for individual carbohydrates were further ascertained by affinity studies using synthetic peptide ligands with immobilized carbohydrates. Binding affinities of the synthetic ligand peptides to lactose, in comparison to the intact full-length lectins, were determined by surface acoustic wave (SAW) biosensor analysis and provided micromolar K D values for the intact lectins, in agreement with results of previous ITC and SPR studies. Binding affinities of the epitope peptides were approximately two orders of magnitude lower, consistent with their smaller size and assembled arrangement in the carbohydrate recognition domains. Graphical Abstract ᅟ.

A novel online comprehensive two-dimensional liquid chromatography (2DLC) coupled with quadrupole time-of-flight (Q-TOF) mass spectrometry (MS) method is developed for the analysis of Curcuma kwangsiensis (C. kwangsiensis) extract. In this system, a newly developed phenyl/tetrazole sulfoether (PTAS) bonded stationary phase was introduced to construct RPLC×RPLC combined with C18. The unique structure endowed PTAS with very different selectivity from C18, reaching a high orthogonality of 93.2%. Moreover, such a combination settled compatibility issues because of the weaker hydrophobic retaining property of PTAS, thus allowing direct interfacing in online configuration. As a result of coupling with the mass spectrometry, a four-dimensional (4D) data plot was presented, in which 439 peaks (containing positive mode and negative mode) were counted, and 105 compounds were grouped and tentatively identified in C. kwangsiensis extract, including 73 unreported ones. Some novel types of compounds with masses exceeding 500 were discovered for the first time. Besides, compared to one-dimensional liquid chromatography (1DLC), the great resolution power of this system allowed separation of more isomers. These results provide supplementary to the material basis of C. Kwangsiensis and in-depth research should be conducted. The configuration of RPLC×RPLC-Q-TOF MS can be a powerful and efficient tool for separation and characterization of chemical substances in complicated herbal extracts., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Penicillins and some non-penicillin β-lactams may cause potentially life-threatening allergic reactions. Thus, possible cross contamination of β-lactams in food or drugs can put people at risk. Therefore, when there is a reasonable possibility that a non-penicillin product could be contaminated by penicillin, the drug products are tested for penicillin contamination. Here, a sensitive and rapid method for simultaneous determination of multiple β-lactam antibiotics using high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated. Mass spectral acquisition was performed on a Q-Exactive HF mass spectrometer in positive ion mode with parallel reaction monitoring (PRM). The method was validated for seven β-lactam antibiotics including one or two from each class and a synthetic intermediate. The quantification precision and accuracy at 200 ppb were in the range of ± 1.84 to ± 4.56 and - 5.20 to 3.44%, respectively. The limit of detection (LOD) was 0.2 ppb, and the limit of quantitation (LOQ) was 2 ppb with a linear dynamic range (LDR) of 2-2000 ppb for all eight β-lactams. From various drug products, the recoveries of eight β-lactams at 200 and 2 ppb ranged from 93.8 ± 3.2 to 112.1 ± 4.2% and 89.7 ± 4.6 to 110.6 ± 1.9%, respectively. The application of the method for detecting cross contamination of trace β-lactams (0.2 ppb) and for monitoring facility surface cleaning was also investigated. This sensitive and fast method was fit-for-purpose for detecting and quantifying trace amount of β-lactam contamination, monitoring cross contamination in manufacturing processes, and determining potency for regulatory purposes and for quality control.

To gain a deep understanding of yeast-cell response to heat stress, multiple laboratory strains have been intensively studied via genome-wide expression analysis for the mechanistic dissection of classical heat-shock response (HSR). However, robust industrial strains of Saccharomyces cerevisiae have hardly been explored in global analysis for elucidation of the mechanism of thermotolerant response (TR) during fermentation. Herein, we employed data-independent acquisition and sequential window acquisition of all theoretical mass spectra based proteomic workflows to characterize proteome remodeling of an industrial strain, ScY01, responding to prolonged thermal stress or transient heat shock. By comparing the proteomic signatures of ScY01 in TR versus HSR as well as the HSR of the industrial strain versus a laboratory strain, our study revealed disparate response mechanisms of ScY01 during thermotolerant growth or under heat shock. In addition, through proteomics data-mining for decoding transcription factor interaction networks followed by validation experiments, we uncovered the functions of two novel transcription factors, Mig1 and Srb2, in enhancing the thermotolerance of the industrial strain. This study has demonstrated that accurate and high-throughput quantitative proteomics not only provides new insights into the molecular basis for complex microbial phenotypes but also pinpoints upstream regulators that can be targeted for improving the desired traits of industrial microorganisms.

Microhaplotypes have become a new type of forensic marker with a great ability to identify and deconvolute mixtures because massively parallel sequencing (MPS) allows the alleles (haplotypes) of the multi-SNP loci to be determined directly for an individual. As originally defined, a microhaplotype locus is a short segment of DNA with two or more SNPs defining three or more haplotypes. The length is short enough, less than about 300 bp, that the read length of current MPS technology can produce a phase-known sequence of each chromosome of an individual. As part of the discovery phase of our studies, data on 130 microhaplotype loci with estimates of haplotype frequency data on 83 populations have been published. To provide a better picture of global allele frequency variation, we have now tested 13 more populations for 65 of the microhaplotype loci from among those with higher levels of inter-population gene frequency variation, including 8 loci not previously published. These loci provide clear distinctions among 6 biogeographic regions and provide some information distinguishing up to 10 clusters of populations.

Comprehensive characterization of the N-glycome of a therapeutic is challenging because glycans may harbor numerous modifications (e.g., phosphorylation, sulfation, sialic acids with possible O-acetylation). The current report presents a comparison of two chromatographic platforms for the comprehensive characterization of a recombinant human erythropoietin (rhEPO) N-glycome. The two platforms include a common workflow based on 2-AB-derivatization and hydrophilic interaction chromatography (HILIC) and a native N-linked glycan workflow employing high performance anion exchange (HPAE) chromatography. Both platforms were coupled to an Orbitrap mass spectrometer, and data dependent HCD fragmentation allowed confident structural elucidation of the glycans. Each platform identified glycans not revealed by the other, and both exhibited strengths and weaknesses. The reductive amination based HILIC workflow provided better throughput and sensitivity, had good isomer resolution, and revealed the presence of O-acetylated sialic acids. However, it exhibited poor performance toward phosphorylated glycans and did not reveal the presence of sulfated glycans. Furthermore, reductive amination introduced dehydration artifacts and modified the glycosylation profile in the rhEPO glycome. Conversely, HPAE provided unbiased charge classification (sialylation levels), improved isomer resolution, and revealed multiple phosphorylated and sulfated structures, but delivered lower throughput, had artifact peaks due to epimer formation, and loss of sialic acid O-acetylation. The MS 2 based identification of phosphorylated and sulfated glycans was not possible in HILIC mode due to their poor solubility caused by the high acetonitrile concentrations employed at the beginning of the gradient. After analyzing the glycome by both approaches and determining the glycans present, a glycan library was created for site specific glycopeptide analyses. Glycopeptide analyses confirmed all the compositions annotated by the combined use of 2-AB- and native glycan workflows and provided site specific location of the glycans. These two platforms were complementary and in combination delivered a more thorough and comprehensive characterization of the rhEPO N-glycome, supporting regulatory conformance for the pharmaceutical industry.

Synthetic biology builds upon the techniques and successes of genetics, molecular biology, and metabolic engineering by applying engineering principles to the design of biological systems. The field still faces substantial challenges, including long development times, high rates of failure, and poor reproducibility. One method to ameliorate these problems would be to improve the exchange of information about designed systems between laboratories. The synthetic biology open language (SBOL) has been developed as a standard to support the specification and exchange of biological design information in synthetic biology, filling a need not satisfied by other pre-existing standards. This document details version 2.2.0 of SBOL that builds upon version 2.1.0 published in last year's JIB special issue. In particular, SBOL 2.2.0 includes improved description and validation rules for genetic design provenance, an extension to support combinatorial genetic designs, a new class to add non-SBOL data as attachments, a new class for genetic design implementations, and a description of a methodology to describe the entire design-build-test-learn cycle within the SBOL data model.

Acoustic cytometry uses radiation pressure forces instead of or in addition to hydrodynamic focusing to position cells or particles in a flowing stream for analysis. Commercial implementations to date combine both hydrodynamic and acoustic focusing together to enable high precision analysis of a broad dynamic range of volumetric sample input rates up to an order of magnitude higher than is practical with hydrodynamic focus alone. This capability allows great flexibility in reducing assay time or modifying or eliminating concentration requirements or concentration steps in sample preparation protocols. It also provides a practical method for processing sub-microliter volumes using sample dilution. In order to take full advantage of this dynamic range, it is necessary to understand the fundamental benefits and limitations of acoustic focusing as applied to flow cytometry. © 2018 by John Wiley & Sons, Inc., (Copyright © 2018 John Wiley & Sons, Inc.)

Sialylated oligosaccharides are important components in mammalian milk. They play a key role in the health and growth of infants by helping to shape up infant's gastrointestinal microbiota and defense against infection by various pathogenic agents. A detailed knowledge of the structures, compositions, and quantities of the sialylated milk oligosaccharides (SMOs) is a prerequisite for understanding their biological roles. However, because of the presence of very large amounts of lactose and neutral oligosaccharides, accurate analysis of SMOs is difficult. A pretreatment step is required to remove lactose and neutral oligosaccharides but conventional off-line pretreatment methods are time-consuming and of poor reproducibility. In this presentation, we linked solid-phase extraction (SPE) with hydrophilic interaction chromatography (HILIC) followed by mass spectrometry (MS) identification for the analysis of SMOs. A SPE column with electrostatic repulsion function was used for removal of lactose and neutral oligosaccharides, a HILIC analytical column for separation of the SMOs, and negative-ion electrospray ionization tandem MS was used for their identification and sequencing. The success of the established online SPE-HILIC-MS method was demonstrated by profiling of SMOs in human to investigate detailed SMO changes during lactation period and in animals to compare the difference in SMO contents among the different species.

Per- and polyfluoroalkyl substances (PFASs) represent a group of synthetic chemicals, and they have quite different physicochemical properties, which result in difficulties of their simultaneous determination in a single injection. A sensitive, reliable, and fully automated method was developed for simultaneously detecting 10 classes of PFASs (total of 43) in human serum using online Turboflow SPE-UHPLC-MS/MS. This method provided high linearity of matrix-matched calibration standards (R > 0.99), excellent method limits of detection (MLODs) (0.013-0.089 ng mL -1 ), satisfactory matrix spiked recoveries (84.3-109%) and relative standard deviations (RSDs) (intra-day RSDs: 1.3-12.6%, inter-day RSDs: 1.7-13.8%, inter-week RSDs: 1.8-13.5%, inter-month RSDs: 3.1-12.4%), short analysis time (19 min per sample) and small sample amount requirement (25 μL), which were suitable for large-scale epidemiologic studies. Moreover, the method provided the feasibility of real-time monitoring for the degradation kinetics of PFASs precursors both in vitro and in vivo. The quality of the present method was further verified by repetitive analysis of a standard reference material (SRM 1957), with the deviations of the targeted PFAS concentrations ranging from 1.9% to 14.2% (n = 5) between the detected and reference values. The present study also determined values for several PFASs in SRM 1957 other than those on the certificate, for the first time, such as N-EtFOSA, 6:2 Cl-PFESA, and PFBA. Finally, the established method was applied to detect PFASs in serum samples of 15 ordinary people and 15 occupational workers, and 6:2 FTSA was found as the dominant precursor., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Current Chinese Pharmacopoeia (ChP) standards apply liquid extraction combined with one dimensional liquid chromatography (1DLC) method for determining alkaloids in herbal medicines. The complex pretreatments lead to a low analytical efficiency and possible component loss. In this study, a heart cutting reversed phase - strong cation exchange two dimensional liquid chromatography (RP - SCX 2DLC) approach was optimized for simultaneously quantifying tropane alkaloids (anisodine, scopolamine and hyoscyamine) in herbal medicines and herbal medicine tablets without further treatment of the filtered extract. The chromatographic conditions were systematically optimized in terms of column type, mobile phase composition and flow rate. To improve peak capacity and obtain symmetric peak shape of alkaloids, a polar group embedded C18 column combined with chaotropic salts was used in the first dimension. To remove the disturbance of non-alkaloids, achieve unique selectivity and acquire symmetric peak shape of alkaloids, an SCX column combined with phosphate buffer was used in the second dimension. Method validation was performed in terms of linearity, precision (0.54-0.82%), recovery (94.1-105.2%), limit of detection (LOD) and limit of quantification (LOQ) of the three analytes varied between 0.067-0.115mgL -1 and 0.195-0.268mgL -1 , respectively. The method demonstrated superiority over 1DLC method in respect of resolution (less alkaloid co-eluted), sample preparation (no pretreatment procedure) and transfer rate (minimum component loss). The optimized RP - SCX 2DLC approach was subsequently applied to quantify target alkaloids in five herbal medicines and herbal medicine tablets from three different manufactures. The results demonstrated that the developed heart cutting RP - SCX 2DLC approach represented a new, strategically significant methodology for the quality evaluation of tropane alkaloid in related herbal medicines that involve complex chemical matrix., (Copyright © 2017. Published by Elsevier B.V.)

As science evolves, the need for more efficient and innovative knowledge transfer capabilities becomes evident. Advances in drug discovery and delivery sciences have directly impacted the pharmaceutical industry, though the added complexities have not shortened the development process. These added complexities also make it difficult for scientists to rapidly and effectively transfer knowledge to offset the lengthened drug development timelines. While webcams, camera phones, and iPads have been explored as potential new methods of real-time information sharing, the non-"hands-free" nature and lack of viewer and observer point-of-view render them unsuitable for the R&D laboratory or manufacturing setting. As an alternative solution, the Microsoft HoloLens mixed-reality headset was evaluated as a more efficient, hands-free method of knowledge transfer and information sharing. After completing a traditional method transfer between 3 R&D sites (Rahway, NJ; West Point, PA and Schnachen, Switzerland), a retrospective analysis of efficiency gain was performed through the comparison of a mock method transfer between NJ and PA sites using the HoloLens. The results demonstrated a minimum 10-fold gain in efficiency, weighing in from a savings in time, cost, and the ability to have real-time data analysis and discussion. In addition, other use cases were evaluated involving vendor and contract research/manufacturing organizations., (Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Objectives: In young children, the use of fecal calprotectin (fCP) as a biomarker is limited because reference values have not been widely accepted up to now. Moreover, reference values for fecal eosinophil-derived neurotoxin (fEDN) in children have not been established. The aim of the present study was to investigate fCP and fEDN levels in young healthy children to establish reference values., Methods: Stool samples were obtained from healthy children ages 0 to 12 years. fCP and fEDN levels were analyzed using the EliA Calprotectin 2 assay (Phadia AB) and a novel research assay (on the ImmunoCAP platform), respectively., Results: In the 174 included children (87 boys), 95th Percentile values ranged from 1519 mg/kg at 0 months to 54.4 mg/kg at 144 months for fCP and from 9.9 mg/kg at 0 months to 0.2 mg/kg at 144 months for fEDN. There was a statistically significant association between age and fCP concentrations (P < 0.001) and age and fEDN concentrations (P < 0.001). We also found a statistically significant association between fEDN and fCP concentrations (rho = 0.52, P < 0.001). According to our results, we provide a nomogram and we suggest 3 different age groups for evaluation of fCP and fEDN concentrations, the 95th percentile being respectively 910.3 and 7.4 mg/kg for 0-12 months, 285.9 and 2.9 mg/kg for >1 to 4 years, and 54.4 and 0.2 mg/kg for >4 to 12 years., Discussion: By using an improved analytical method, we have confirmed that young healthy children have higher fCP concentrations than healthy adults. We, for the first time, report reference values for fEDN concentrations in a pediatric population. The proposed nomograms and reference values for fCP and fEDN are aimed at facilitating the applicability of biomarkers for both neutrophil- and eosinophil-mediated intestinal inflammation in children in clinical practice.

Background: Among septic patients admitted to the intensive care unit (ICU), early recognition of those with the highest risk of death is of paramount importance. We evaluated the prognostic value of Procalcitonin (PCT), mid regional-proadrenomedullin (MR-proADM), copeptine and CT-proendothelin 1 (CT-ProET 1) concentrations., Methods: This was a prospective cohort study, which included 173 septic patient admitted to one ICU. Blood samples for biomarker measurements were obtained upon admission and on day 5. The predictive value of each biomarker regarding the risk of death at day 28 was assessed. The fluid balance was evaluated from admission to day 5., Results: All cause ICU mortality was 36.4%. All the biomarkers except CT-ProET-1 were significantly more elevated in the non-survivors than in the survivors upon day 1. This was especially true for MR-proADM (8.6 [5.9] vs. 4.4 [3.9] nmol/L; P < 0.0001) and for the CT-proET-1/MR-proADM ratio (52.9 [22.4] vs. 31.3 [26.6] arbitrary units; P < 0.0001). The best AUROCC values on day 1 were obtained with MR-ProADM and the CT-proET-1/MR-proADM ratio as well (0.75 [0.67-0.85] and 0.82 [0.75-0.89]; 95% CI, respectively). An improved accuracy was achieved on day 5. Moreover, MR-ProADM baseline levels and fluid balance over the 5-day period following ICU admission were strongly correlated (Rho = 0.41; P < 0.001)., Conclusions: In patients admitted to the ICU with sepsis, MR-ProADM on admission was the best predictor of short-term clinical outcome if compared with others. This could be related to its ability to predict fluid sequestration.

Digital PCR, a state-of-the-art nucleic acid quantification technique, works by spreading the target material across a large number of partitions. The average number of molecules per partition is estimated using Poisson statistics, and then converted into concentration by dividing by partition volume. In this standard approach, identical partition sizing is assumed. Violations of this assumption result in underestimation of target quantity, when using Poisson modeling, especially at higher concentrations. The Poisson-Plus Model accommodates for this underestimation, if statistics of the volume variation are well characterized. The volume variation was measured on the chip array based QuantStudio 3D Digital PCR System using the ROX fluorescence level as a proxy for effective load volume per through-hole. Monte Carlo simulations demonstrate the efficacy of the proposed correction. Empirical measurement of model parameters characterizing the effective load volume on QuantStudio 3D Digital PCR chips is presented. The model was used to analyze digital PCR experiments and showed improved accuracy in quantification. At the higher concentrations, the modeling must take effective fill volume variation into account to produce accurate estimates. The extent of the difference from the standard to the new modeling is positively correlated to the extent of fill volume variation in the effective load of your reactions.

In the present study, we report the first silver-dependent enantiodivergent gold-catalysed reaction. The asymmetric intramolecular hydroamination of alkenes catalysed by the combination of a single chiral binuclear gold(I) chloride complex and silver perchlorate can afford both enantiomers of the products by a simple solvent change from toluene to methanol. Such an enantiodivergent reaction is strictly independent of the reaction temperature or of the nature of the catalyst anion and displays the same first-order kinetic rate law with respect to substrate concentration in both solvents. Beyond a simple solvent effect the enantioinversion is controlled by gold-silver chloride adducts which occur only in methanol and allow a dual activation of the reagent. While one single gold atom activates the alkene moiety, the other gold atom forms an oxophilic gold-silver chloride adduct which is likely to interact with the carbamate function. By comparison with toluene, which affords (S)-enantiomer, this proximal and bimetallic activation would allow an opposite stereodifferentiation of the two diastereomeric intermediates during the final protodeauration step and lead therefore to the (R)-enantiomer., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In-depth and reproducible protein measurement in many biological samples is often critical for pharmaceutical/biomedical proteomics but remains challenging. MS1-based quantification using quadrupole/ultrahigh-field Orbitrap (Q/UHF-Orbitrap) holds great promise, but the critically important experimental approaches enabling reliable large-cohort analysis have long been overlooked. Here we described an IonStar experimental strategy achieving excellent quantitative quality of MS1 quantification. Key features include: (i) an optimized, surfactant-aided sample preparation approach provides highly efficient (>75% recovery) and reproducible (<15% CV) peptide recovery across large cell/tissue cohorts; (ii) a long column with modest gradient length (2.5 h) yields the optimal balance of depth/throughput on a Q/UHF-Orbitrap; (iii) a large-ID trap not only enables highly reproducible gradient delivery as for the first time observed via real-time conductivity monitoring, but also increases quantitative loading capacity by >8-fold and quantified >25% more proteins; (iv) an optimized HCD-OT markedly outperforms HCD-IT when analyzing large cohorts with high loading amounts; (v) selective removal of hydrophobic/hydrophilic matrix components using a novel selective trapping/delivery approach enables reproducible, robust LC-MS analysis of >100 biological samples in a single set, eliminating batch effect; (vi) MS1 acquired at higher resolution (fwhm = 120 k) provides enhanced S/N and quantitative accuracy/precision for low-abundance species. We examined this pipeline by analyzing a 5 group, 20 samples biological benchmark sample set, and quantified 6273 unique proteins (≥2 peptides/protein) under stringent cutoffs without fractionation, 6234 (>99.4%) without missing data in any of the 20 samples. The strategy achieved high quantitative accuracy (3-6% media error), low intragroup variation (6-9% media intragroup CV) and low false-positive biomarker discovery rates (3-8%) across the five groups, with quantified protein abundances spanning >6.5 orders of magnitude. Finally, this strategy is straightforward, robust, and broadly applicable in pharmaceutical/biomedical investigations.

Today the primary DNA markers used in forensics are short tandem repeat (STR) polymorphisms (STRPs), initially selected because they are highly polymorphic. However, the increasingly common need to deal with samples with a mixture of DNA from two or more individuals sometimes is complicated by the inherent stutter involved with PCR amplification, especially in strongly unbalanced mixtures when the minor component coincides with the stutter range of the major component. Also, the STRPs in use provide little evidence of ancestry of a single source sample beyond broad "continental" resolution. Methodologies for analyzing DNA have become much more powerful in recent years. Massively parallel sequencing (MPS) is a new method being considered for routine use in forensics. Primarily to aid in mixture deconvolution and avoid the issue of stutter, we have begun to investigate a new type of forensic marker, microhaplotype loci, that will provide useful information on mixtures of DNA and on ancestry when typed using massively parallel sequencing (MPS). We have identified 130 loci and estimated their haplotype (allele) frequencies in 83 different population samples. Many of these loci are shown to be highly informative for individual identification and for mixture identification and deconvolution., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)