Entanglement in the quantum phases of an unfrustrated Rydberg atom array.

Recent experimental advances have stimulated interest in the use of large, two-dimensional arrays of Rydberg atoms as a platform for quantum information processing and to study exotic many-body quantum states. However, the native long-range interactions between the atoms complicate experimental analysis and precise theoretical understanding of these systems. Here we use new tensor network algorithms capable of including all long-range interactions to study the ground state phase diagram of Rydberg atoms in a geometrically unfrustrated square lattice array. We find a greatly altered phase diagram from earlier numerical and experimental studies, revealed by studying the phases on the bulk lattice and their analogs in experiment-sized finite arrays. We further describe a previously unknown region with a nematic phase stabilized by short-range entanglement and an order from disorder mechanism. Broadly our results yield a conceptual guide for future experiments, while our techniques provide a blueprint for converging numerical studies in other lattices.

Optimised plasma sample preparation and LC-MS analysis to support large-scale proteomic analysis of clinical trial specimens: Application to the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) trial.

PURPOSE: Robust, affordable plasma proteomic biomarker workflows are needed for large-scale clinical studies. We evaluated aspects of sample preparation to allow liquid chromatography-mass spectrometry (LC-MS) analysis of more than 1500 samples from the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) trial of adults with type 2 diabetes. METHODS: Using LC-MS with data-independent acquisition we evaluated four variables: plasma protein depletion, EDTA or citrated anti-coagulant blood collection tubes, plasma lipid depletion strategies and plasma freeze-thaw cycles. Optimised methods were applied in a pilot study of FIELD participants. RESULTS: LC-MS of undepleted plasma conducted over a 45 min gradient yielded 172 proteins after excluding immunoglobulin isoforms. Cibachrome-blue-based depletion yielded additional proteins but with cost and time expenses, while immunodepleting albumin and IgG provided few additional identifications. Only minor variations were associated with blood collection tube type, delipidation methods and freeze-thaw cycles. From 65 batches involving over 1500 injections, the median intra-batch quantitative differences in the top 100 proteins of the plasma external standard were less than 2%. Fenofibrate altered seven plasma proteins. CONCLUSIONS AND CLINICAL RELEVANCE: A robust plasma handling and LC-MS proteomics workflow for abundant plasma proteins has been developed for large-scale biomarker studies that balance proteomic depth with time and resource costs.

Viral Biomarker Detection and Validation Using MALDI Mass Spectrometry Imaging (MSI).

(1) Background: MALDI imaging is a technique that still largely depends on time of flight (TOF)-based instrument such as the Bruker UltrafleXtreme. While capable of performing targeted MS/MS, these instruments are unable to perform fragmentation while imaging a tissue section necessitating the reliance of MS1 values for peptide level identifications. With this premise in mind, we have developed a hybrid bioinformatic/image-based method for the identification and validation of viral biomarkers. (2) Methods: Formalin-Fixed Paraffin-Embedded (FFPE) mouse samples were sectioned, mounted and prepared for mass spectrometry imaging using our well-established methods. Peptide identification was achieved by first extracting confident images corresponding to theoretical viral peptides. Next, those masses were used to perform a Peptide Mmass Fingerprint (PMF) searched against known viral FASTA sequences against a background mouse FASTA database. Finally, a correlational analysis was performed with imaging data to confirm pixel-by-pixel colocalization and intensity of viral peptides. (3) Results: 14 viral peptides were successfully identified with significant PMF Scores and a correlational result of >0.79 confirming the presence of the virus and distinguishing it from the background mouse proteins. (4) Conclusions: this novel approach leverages the power of mass spectrometry imaging and provides confident identifications for viral proteins without requiring MS/MS using simple MALDI Time Of Flight/Time Of Flight (TOF/TOF) instrumentation.

OXSR1 inhibits inflammasome activation by limiting potassium efflux during mycobacterial infection.

Pathogenic mycobacteria inhibit inflammasome activation to establish infection. Although it is known that potassium efflux is a trigger for inflammasome activation, the interaction between mycobacterial infection, potassium efflux, and inflammasome activation has not been investigated. Here, we use Mycobacterium marinum infection of zebrafish embryos and Mycobacterium tuberculosis infection of THP-1 cells to demonstrate that pathogenic mycobacteria up-regulate the host WNK signalling pathway kinases SPAK and OXSR1 which control intracellular potassium balance. We show that genetic depletion or inhibition of OXSR1 decreases bacterial burden and intracellular potassium levels. The protective effects of OXSR1 depletion are at least partially mediated by NLRP3 inflammasome activation, caspase-mediated release of IL-1ß, and downstream activation of protective TNF-α. The elucidation of this druggable pathway to potentiate inflammasome activation provides a new avenue for the development of host-directed therapies against intracellular infections.

Proteomic Analysis of Whole Blood Using Volumetric Absorptive Microsampling for Precision Medicine Biomarker Studies.

Microsampling of patient blood promises several benefits over conventional phlebotomy practices to facilitate precision medicine studies. These include at-home patient blood collection, supporting telehealth monitoring, minimal postcollection processing, and compatibility with nonrefrigerated transport and storage. However, for proteomic biomarker studies, mass spectrometry of whole blood has generally been avoided in favor of using plasma or serum obtained from venepuncture. We evaluated the use of a volumetric absorptive microsampling (VAMS) device as a sample preparation matrix to enable LC-MS proteomic analyses of dried whole blood. We demonstrated the detection and robust quantitation of up to 1600 proteins from single-shot shotgun-LC-MS analysis of dried whole blood, greatly enhancing proteome depth compared with conventional single-shot LC-MS analyses of undepleted plasma. Some proteins not previously reported in blood were detected using this approach. Various washing reagents were used to demonstrate that proteins can be preferentially removed from VAMS devices prior to downstream analyses. We provide a demonstration that archival frozen blood cell pellets housed under long-term storage (exceeding 5 years) are compatible with VAMS to enable quantitation of potential biomarker proteins from biobank repositories. These demonstrations are important steps in establishing viable analysis workflows to underpin large-scale precision medicine studies. Data are available via ProteomeXchange with the identifier PXD028605.

Matrix phase fractionation: Investigating the compromise between dynamic range of analyte extraction and spatial resolution in mass spectrometry imaging.

RATIONALE: Matrix-assisted laser desorption ionisation with mass spectrometry imaging (MSI) has seen rapid development in recent years and as such is becoming an important technique for the mapping of biomolecules from the surface of tissues. One key area of development is the optimisation of analyte extraction by using modified matrices or mixes of common ones. METHODS: A series of serial sections were prepared for lipid MSI by either dry coating (sublimation) or by wet spray application of several matrices. These samples were then evaluated for analyte extraction, delocalisation and dynamic range. RESULTS: We have shown that the spraying and sublimation methods of matrix application can be used complementarily. This creates large datasets, with each preparation method applied narrowly and then interpreted as a 'fraction' of the whole. Once combined, the dynamic range is significantly increased. We have dubbed this technique 'matrix phase fractionation'. CONCLUSIONS: We have found that, by utilising matrix phase fractionation for the detection of lipids in brain tissue, it is possible to create a significantly more comprehensive dataset than would otherwise be possible with traditional 'single-run' workflows.

Association of Use of the NEJM Knowledge+ Product and Performance on the ABIM IM-MOC Exam.

Phenomenon: Internal medicine physicians in the United States must pass the American Board of Internal Medicine Internal Medicine Maintenance of Certification (ABIM IM-MOC) examination as part of their ABIM IM-MOC requirements. Many of these physicians use an examination product to help them prepare, such as e-Learning products, including the ACP's MKSAP, UpToDate, and NEJM Knowledge+, yet their effectiveness remains largely unstudied. Approach: We compared ABIM IM-MOC examination performance among 177 physicians who attempted an ABIM IM-MOC examination between 2014-2017 and completed at least 75% of the NEJM Knowledge+ product prior to the ABIM IM-MOC examination and 177 very similar matched control physicians who did not use NEJM Knowledge+. Our measures of ABIM IM-MOC exam performance for NEJM Knowledge+ users were based on the results of the first attempt immediately following the NEJM Knowledge+ use and for non-users were based on the applicable matched examination performance. The three dichotomous examination performance outcomes measured on the first attempt at the ABIM IM-MOC examination included pass rate, scoring in the upper quartile, and scoring in the lower quartile. Findings: Use of NEJM Knowledge+ was associated with a regression adjusted 10.6% (5.37% to 15.8%) greater likelihood of passing the MOC examination (p < .001), 10.7% (2.61% to 18.7%) greater likelihood of having an examination score in the top quartile (p = .009), and -10.8% (-16.8% to -4.86%) lower likelihood of being in the bottom quartile of the MOC examination (p < .001) as compared to similar physicians who did not use NEJM Knowledge+. Insight: Physicians who used NEJM Knowledge+ had better ABIM IM-MOC exam performance. Further research is needed to determine what aspects of e-Learning products best prepare physicians for MOC examinations.

Data independent acquisition of plasma biomarkers of response to neoadjuvant chemotherapy in pancreatic ductal adenocarcinoma.

The detection of disease-related plasma biomarkers has challenged the proteomic community for years. Attractive features for plasma proteomics includes the ease of collection and small volume needed for analysis, but on the other hand, the presence of highly abundant proteins complicates sample preparation procedures and reduces dynamic range. Data independent acquisition label free quantitation (DIA-LFQ) by mass spectrometry partly overcomes the dynamic range issue; however, generating the peptide spectral reference libraries that allow extensive analysis of the plasma proteome can be a slow and expensive task which is unattainable for many laboratories. We investigated the re-purposing of publically available plasma proteome datasets and the impact on peptide/protein detection for DIA-LFQ. We carried out these studies in the context of identifying putative biomarkers of response to neoadjuvant chemotherapy (NAC) for pancreatic ductal adenocarcinoma, as no useful plasma biomarkers have been clinically adopted. We demonstrated the benefit in searching DIA data against multiple spectral libraries to show that complement proteins were linked to NAC response in PDAC patients, confirming previous observations of the prognostic utility of complement following adjuvant chemotherapy. Our workflow demonstrates that DIA-LFQ can be readily applied in the oncology setting for the putative assignment of clinically relevant plasma biomarkers. STATEMENT OF SIGNIFICANCE: The proteomic mass spectrometry analysis of undepleted, unfractionated human plasma has benefits for sample throughput but remains challenging to obtain deep coverage. This work evaluated the re-purposing of open source peptide mass spectrometry data from human plasma to create spectral reference libraries for use in Data independent acquisition (DIA). We showed how seeding in locally acquired data to integrate iRT peptides into spectral libraries increased identification confidence by facilitating querying of multiple libraries. This workflow was applied to the discovery of putative plasma biomarkers for response to neoadjuvant chemotherapy (NAC) in pancreatic ductal adenocarcinoma patients. There is a paucity of prior information in the literature on this topic and we show that good responder patients have reduced levels of complement proteins.

Quantitative Proteomic Profiling of Small Molecule Treated Mesenchymal Stem Cells Using Chemical Probes.

The differentiation of human adipose derived stem cells toward a neural phenotype by small molecules has been a vogue topic in the last decade. The characterization of the produced cells has been explored on a broad scale, examining morphological and specific surface protein markers; however, the lack of insight into the expression of functional proteins and their interactive partners is required to further understand the extent of the process. The phenotypic characterization by proteomic profiling allows for a substantial in-depth analysis of the molecular machinery induced and directing the cellular changes through the process. Herein we describe the temporal analysis and quantitative profiling of neural differentiating human adipose-derived stem cells after sub-proteome enrichment using a bisindolylmaleimide chemical probe. The results show that proteins enriched by the Bis-probe were identified reproducibly with 133, 118, 126 and 89 proteins identified at timepoints 0, 1, 6 and 12, respectively. Each temporal timepoint presented several shared and unique proteins relative to neural differentiation and their interactivity. The major protein classes enriched and quantified were enzymes, structural and ribosomal proteins that are integral to differentiation pathways. There were 42 uniquely identified enzymes identified in the cells, many acting as hubs in the networks with several interactions across the network modulating key biological pathways. From the cohort, it was found by gene ontology analysis that 18 enzymes had direct involvement with neurogenic differentiation.

Reporting of Hybrid Data and the Difficulties with Cross-Discipline Research Techniques.

Peer review is the way in which we, as scientists, criticise, check, and confirm the findings of our colleagues. The process of peer review relies on individuals in all fields applying their particular expertise and determining if they agree with the findings submitted for publication. In recent years, there has been a significant rise in the number of manuscripts submitted for publication that draw from a range of disparate and complementary fields. This has created the curious situation where an expert may be requested to review a manuscript that is only partially within their immediate field of expertise. The issue that arises is that, without full knowledge of the data, techniques, methodologies, and principles that are presented, it is difficult for reviewers to make properly informed decisions, especially when it can take an entire career to reach that specific level of expertise in a single field. From this perspective, we explore these issues and also provide a commentary on how peer review could evolve in the context of a changing cross-disciplinarily-focused scientific landscape.

Exploring the Magnetic Properties of the Largest Single-Molecule Magnets.

The giant {Mn70} and {Mn84} wheels are the largest nuclearity single-molecule magnets synthesized to date, and understanding their magnetic properties poses a challenge to theory. Starting from first-principles calculations, we explore the magnetic properties and excitations in these wheels using effective spin Hamiltonians. We find that the unusual geometry of the superexchange pathways leads to weakly coupled {Mn7} subunits carrying an effective S = 2 spin. The spectrum exhibits a hierarchy of energy scales and massive degeneracies, with the lowest-energy excitations arising from Heisenberg-ring-like excitations of the {Mn7} subunits around the wheel. We further describe how weak longer-range couplings can select the precise spin ground-state of the Mn wheels out of the nearly degenerate ground-state band.

Murine and related chapparvoviruses are nephro-tropic and produce novel accessory proteins in infected kidneys.

Mouse kidney parvovirus (MKPV) is a member of the provisional genus Chapparvovirus that causes renal disease in immune-compromised mice, with a disease course reminiscent of polyomavirus-associated nephropathy in immune-suppressed kidney transplant patients. Here we map four major MKPV transcripts, created by alternative splicing, to a common initiator region, and use mass spectrometry to identify "p10" and "p15" as novel chapparvovirus accessory proteins produced in MKPV-infected kidneys. p15 and the splicing-dependent putative accessory protein NS2 are conserved in all near-complete amniote chapparvovirus genomes currently available (from mammals, birds and a reptile). In contrast, p10 may be encoded only by viruses with >60% amino acid identity to MKPV. We show that MKPV is kidney-tropic and that the bat chapparvovirus DrPV-1 and a non-human primate chapparvovirus, CKPV, are also found in the kidneys of their hosts. We propose, therefore, that many mammal chapparvoviruses are likely to be nephrotropic.

An Inexpensive, simple calibration method for MALDI TOF/TOF systems.

The array of analytes that can be measured by MADLI MS has created an equally vast range of calibration mixtures. The inherent problem with this is that acquiring all of them at commercial rates can be prohibitively expensive. With this in mind, we have created a low-cost alternative to the most commonly used peptide calibrants. We were able to achieve an overall 78 ppm mass accuracy across a mass range of 900 to 2500 Da which was comparable to the mass accuracy achievable with commercial peptide mixes and hence has become a viable alternative.

What is Normalization? The Strategies Employed in Top-Down and Bottom-Up Proteome Analysis Workflows.

The accurate quantification of changes in the abundance of proteins is one of the main applications of proteomics. The maintenance of accuracy can be affected by bias and error that can occur at many points in the experimental process, and normalization strategies are crucial to attempt to overcome this bias and return the sample to its regular biological condition, or normal state. Much work has been published on performing normalization on data post-acquisition with many algorithms and statistical processes available. However, there are many other sources of bias that can occur during experimental design and sample handling that are currently unaddressed. This article aims to cast light on the potential sources of bias and where normalization could be applied to return the sample to its normal state. Throughout we suggest solutions where possible but, in some cases, solutions are not available. Thus, we see this article as a starting point for discussion of the definition of and the issues surrounding the concept of normalization as it applies to the proteomic analysis of biological samples. Specifically, we discuss a wide range of different normalization techniques that can occur at each stage of the sample preparation and analysis process.

NEJM Knowledge+ Question of the Week: A Novel Virtual Learning Community Effectively Utilizing an Online Discussion Forum.

Objectives: Asynchronous virtual learning communities provide learners with the ability to enhance their learning and contribute to their peers' learning in a safe environment. However, the tone and content of learner comments, the level of engagement among learners, and the role of moderators have not been well studied within non-course-related virtual learning communities. Therefore, we sought to explore these characteristics using the NEJM Knowledge+ Question of the Week (NEJM Knowledge+ QoW) forum, a web-based asynchronous virtual learning community. Methods: We reviewed 73 NEJM Knowledge+ questions posted on the QoW forum between 2015 and 2016. We then selected three QoWs to analyze through a multistep coding process based on three broad criteria that aligned with our study aims. Results: Learner comments reflected both positive and critical tones, with learners sharing their own clinical practice and local experiences to contextualize their perspectives and reactions to both the QoW answer and the responses of other learners. Learners also commonly requested moderators to act as expert referees. Conclusion: Asynchronous virtual learning communities can engage learners by providing the opportunity to enhance their knowledge through responding to proposed medical scenarios and sharing their experiences in a discussion forum. Future work should examine the impact that geographic region has on asynchronous virtual learning communities and the role of moderators in shaping the learning experience.

Higher Mass Accuracy MALDI-TOF/TOF Lipid Imaging of Human Brain Tissue in Alzheimer's Disease.

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) is a well-established technique for elucidating the location and relative abundance of a range of biomolecules. More recently, research into this technique has shifted from simple discovery and demonstration of utility to application in biomedical research. Here, we describe a protocol utilizing MALDI-IMS for the spatial mapping of lipids in brain tissue from normal human brains and brains from patients with Alzheimer's disease, in the context of Alzheimer's disease. Improved accuracy calibration of the instrument from the tissue surface is emphasized, as this allows for significantly improved mass determination in time of flight (TOF)-based instruments enabling more confident preliminary lipid identification. This improved initial result allows MALDI-IMS data to be complemented with additional instrumentation, such as liquid chromatography mass spectrometry workflows or specialized non-TOF systems such as Fourier transform cyclotron resonance instruments. This method is not limited to human tissue and can be applied to virtually any lipid-rich formalin-fixed tissue. © 2019 by John Wiley & Sons, Inc.

Identification of Novel Natural Substrates of Fibroblast Activation Protein-alpha by Differential Degradomics and Proteomics.

Fibroblast activation protein-alpha (FAP) is a cell-surface transmembrane-anchored dimeric protease. This unique, constitutively active serine protease has both dipeptidyl aminopeptidase and endopeptidase activities and can hydrolyze the post-proline bond. FAP expression is very low in adult organs but is upregulated by activated fibroblasts in sites of tissue remodeling, including fibrosis, atherosclerosis, arthritis and tumors. To identify the endogenous substrates of FAP, we immortalized primary mouse embryonic fibroblasts (MEFs) from FAP gene knockout embryos and then stably transduced them to express either enzymatically active or inactive FAP. The MEF secretomes were then analyzed using degradomic and proteomic techniques. Terminal amine isotopic labeling of substrates (TAILS)-based degradomics identified cleavage sites in collagens, many other extracellular matrix (ECM) and associated proteins, and lysyl oxidase-like-1, CXCL-5, CSF-1, and C1qT6, that were confirmed in vitro In addition, differential metabolic labeling coupled with quantitative proteomic analysis also implicated FAP in ECM-cell interactions, as well as with coagulation, metabolism and wound healing associated proteins. Plasma from FAP-deficient mice exhibited slower than wild-type clotting times. This study provides a significant expansion of the substrate repertoire of FAP and provides insight into the physiological and potential pathological roles of this enigmatic protease.

An Atypical Parvovirus Drives Chronic Tubulointerstitial Nephropathy and Kidney Fibrosis.

The occurrence of a spontaneous nephropathy with intranuclear inclusions in laboratory mice has puzzled pathologists for over 4 decades, because its etiology remains elusive. The condition is more severe in immunodeficient animals, suggesting an infectious cause. Using metagenomics, we identify the causative agent as an atypical virus, termed "mouse kidney parvovirus" (MKPV), belonging to a divergent genus of Parvoviridae. MKPV was identified in animal facilities in Australia and North America, is transmitted via a fecal-oral or urinary-oral route, and is controlled by the adaptive immune system. Detailed analysis of the clinical course and histopathological features demonstrated a stepwise progression of pathology ranging from sporadic tubular inclusions to tubular degeneration and interstitial fibrosis and culminating in renal failure. In summary, we identify a widely distributed pathogen in laboratory mice and establish MKPV-induced nephropathy as a new tool for elucidating mechanisms of tubulointerstitial fibrosis that shares molecular features with chronic kidney disease in humans.

The Effect of Collimating Lens Focusing on Laser Beam Shape in Matrix Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS).

Tissue imaging using matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a well-established technique that, in recent years, has seen wider adoption and novel application. Applications such imaging mass spectrometry (IMS) and biotyping are beginning to gain greater exposure and use; however, with limitations in optimization methods, producing the best result often relies on the ability to customize the physical characteristics of the instrumentation, a task that is challenging for most mass spectrometry laboratories. With this in mind, we have described the effect of making simple adjustments to the laser optics at the final collimating lens area, to adjust the laser beam size and shape in order to allow greater customization of the instrument for improving techniques such as IMS. We have therefore been able to demonstrate that improvements can be made without requiring the help of an electrical engineer or external funding in a way that only costs a small amount of time. Graphical Abstract ᅟ.

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows.

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.