Label-Free Profiling of up to 200 Single-Cell Proteomes per Day Using a Dual-Column Nanoflow Liquid Chromatography Platform.

Single-cell proteomics (SCP) has great potential to advance biomedical research and personalized medicine. The sensitivity of such measurements increases with low-flow separations (<100 nL/min) due to improved ionization efficiency, but the time required for sample loading, column washing, and regeneration in these systems can lead to low measurement throughput and inefficient utilization of the mass spectrometer. Herein, we developed a two-column liquid chromatography (LC) system that dramatically increases the throughput of label-free SCP using two parallel subsystems to multiplex sample loading, online desalting, analysis, and column regeneration. The integration of MS1-based feature matching increased proteome coverage when short LC gradients were used. The high-throughput LC system was reproducible between the columns, with a 4% difference in median peptide abundance and a median CV of 18% across 100 replicate analyses of a single-cell-sized peptide standard. An average of 621, 774, 952, and 1622 protein groups were identified with total analysis times of 7, 10, 15, and 30 min, corresponding to a measurement throughput of 206, 144, 96, and 48 samples per day, respectively. When applied to single HeLa cells, we identified nearly 1000 protein groups per cell using 30 min cycles and 660 protein groups per cell for 15 min cycles. We explored the possibility of measuring cancer therapeutic targets with a pilot study comparing the K562 and Jurkat leukemia cell lines. This work demonstrates the feasibility of high-throughput label-free single-cell proteomics.

Design and Characterization of Metal Nanoparticle Infiltrated Mesoporous Metal-Organic Frameworks.

The infiltration of palladium and platinum nanoparticles (NPs) into the mesoporous metal-organic framework (MOF) CYCU-3 through chemical vapor infiltration (CVI) and incipient wetness infiltration (IWI) processes was systematically explored as a means to design novel NP@MOF composite materials for potential hydrogen storage applications. We employed a traditional CVI process and a new ″green″ IWI process using methanol for precursor infiltration and reduction under mild conditions. Transmission electron microscopy-based direct imaging techniques combined with synchrotron-based powder diffraction (SPD), energy-dispersive X-ray spectroscopy, and physisorption analysis reveal that the resulting NP@MOF composites combine key NP and MOF properties. Room temperature hydrogen adsorption capacities of 0.95 and 0.20 mmol/g at 1 bar and 2.9 and 1.8 mmol/g at 100 bar are found for CVI and IWI samples, respectively. Hydrogen spillover and/or physisorption are proposed as the dominating adsorption mechanisms depending on the NP infiltration method. Mechanistic insights were obtained through the crystallographic means using SPD-based difference envelope density analysis, providing previously underexplored details on NP@MOF preparations. Consequently, important host-guest correlations influencing the global hydrogen adsorption properties are discussed, and they demonstrate that employing MOFs as platforms for NPs is an alternative approach to the development of versatile materials for improving current hydrogen storage technologies.

In vivo-generated thrombin and plasmin do not activate the complement system in baboons.

Sepsis concurrently activates both coagulation and complement systems. Although complement activation by bacteria is well documented, work in mice and in vitro suggests that coagulation proteases can directly cleave complement proteins. We aimed to determine whether generation of coagulation proteases in vivo can activate the complement cascade in 2 highly coagulopathic models. We compared temporal changes in activation biomarkers of coagulation (thrombin-antithrombin [TAT]), fibrinolysis (plasmin-antiplasmin [PAP]), and complement (C3b, C5a, C5b-9) in baboons infused with factor Xa (FXa) and phospholipids (FXa/phosphatidylcholine-phosphatidylserine [PCPS]) vs LD100 Escherichia coli We found that, albeit with different timing, both FXa/PCPS and E coli infusion led to robust thrombin and plasmin generation. Conversely, only E coli challenge activated the complement system, reaching a maximum at 2 hours postchallenge during the peaks of lipopolysaccharide and bacteremia but not of TAT and PAP. Despite inducing a strong burst of thrombin and plasmin, FXa/PCPS infusion did not produce measurable levels of complement activation in vivo. Similarly, ex vivo incubation of baboon serum with thrombin, plasmin, or FXa did not show noticeable complement cleavage unless supraphysiologic amounts of enzymes were used. Our results suggest that in vivo-generated thrombin and plasmin do not directly activate the complement in nonhuman primates.

Sphingosine 1-phosphate and its carrier apolipoprotein M in human sepsis and in Escherichia coli sepsis in baboons.

Sphingosine 1-phosphate (S1P) is an important regulator of vascular integrity and immune cell migration, carried in plasma by high-density lipoprotein (HDL)-associated apolipoprotein M (apoM) and by albumin. In sepsis, the protein and lipid composition of HDL changes dramatically. The aim of this study was to evaluate changes in S1P and its carrier protein apoM during sepsis. For this purpose, plasma samples from both human sepsis patients and from an experimental Escherichia coli sepsis model in baboons were used. In the human sepsis cohort, previously studied for apoM, plasma demonstrated disease-severity correlated decreased S1P levels, the profile mimicking that of plasma apoM. In the baboons, a similar disease-severity dependent decrease in plasma levels of S1P and apoM was observed. In the lethal E. coli baboon sepsis, S1P decreased already within 6-8 hrs, whereas the apoM decrease was seen later at 12-24 hrs. Gel filtration chromatography of plasma from severe human or baboon sepsis on Superose 6 demonstrated an almost complete loss of S1P and apoM in the HDL fractions. S1P plasma concentrations correlated with the platelet count but not with erythrocytes or white blood cells. The liver mRNA levels of apoM and apoA1 decreased strongly upon sepsis induction and after 12 hr both were almost completely lost. In conclusion, during septic challenge, the plasma levels of S1P drop to very low levels. Moreover, the liver synthesis of apoM decreases severely and the plasma levels of apoM are reduced. Possibly, the decrease in S1P contributes to the decreased endothelial barrier function observed in sepsis.

Complement inhibition decreases early fibrogenic events in the lung of septic baboons.

Acute respiratory distress syndrome (ARDS) induced by severe sepsis can trigger persistent inflammation and fibrosis. We have shown that experimental sepsis in baboons recapitulates ARDS progression in humans, including chronic inflammation and long-lasting fibrosis in the lung. Complement activation products may contribute to the fibroproliferative response, suggesting that complement inhibitors are potential therapeutic agents. We have been suggested that treatment of septic baboons with compstatin, a C3 convertase inhibitor protects against ARDS-induced fibroproliferation. Baboons challenged with 10(9) cfu/kg (LD50) live E. coli by intravenous infusion were treated or not with compstatin at the time of challenge or 5 hrs thereafter. Changes in the fibroproliferative response at 24 hrs post-challenge were analysed at both transcript and protein levels. Gene expression analysis showed that sepsis induced fibrotic responses in the lung as early as 24 hrs post-bacterial challenge. Immunochemical and biochemical analysis revealed enhanced collagen synthesis, induction of profibrotic factors and increased cell recruitment and proliferation. Specific inhibition of complement with compstatin down-regulated sepsis-induced fibrosis genes, including transforming growth factor-beta (TGF-ß), connective tissue growth factor (CTGF), tissue inhibitor of metalloproteinase 1 (TIMP1), various collagens and chemokines responsible for fibrocyte recruitment (e.g. chemokine (C-C motif) ligand 2 (CCL2) and 12 (CCL12)). Compstatin decreased the accumulation of myofibroblasts and proliferating cells, reduced the production of fibrosis mediators (TGF-ß, phospho-Smad-2 and CTGF) and inhibited collagen deposition. Our data demonstrate that complement inhibition effectively attenuates collagen deposition and fibrotic responses in the lung after severe sepsis. Inhibiting complement could prove an attractive strategy for preventing sepsis-induced fibrosis of the lung.

Mid-infrared multi-mode absorption spectroscopy using interband cascade lasers for multi-species sensing.

An interband cascade laser (ICL) operating at 3.7 µm has been used to perform multimode absorption spectroscopy, MUMAS, at scan rates up to 10 kHz. Line widths of individual modes in the range 10-80 MHz were derived from isolated lines in the MUMAS signatures of HCl. MUMAS data for methane covering a spectral range of 30 nm yielded a detection level of 30 µbar·m for 1 s measurement time at 100 Hz. Simultaneous detection of methane, acetylene, and formaldehyde in a gas mixture containing all three species is reported.

Fatigue, psychological and cognitive impairment following transient ischaemic attack and minor stroke: a systematic review.

Transient ischaemic attack (TIA) and minor stroke are characterized by short-lasting symptoms; however, anecdotal and empirical evidence suggests that these patients experience ongoing cognitive/psychological impairment for which they are not routinely treated. The aims were (i) to investigate the prevalence and time course of fatigue, anxiety, depression, post-traumatic stress disorder(PTSD) and cognitive impairment following TIA/minor stroke; (ii) to explore the impact on quality of life (QoL), change in emotions and return to work; and (iii) to identify where further research is required and potentially inform an intervention study. A systematic review of MEDLINE, EMBASE, PSYCINFO, CINAHL, the Cochrane libraries and the grey literature between January 1993 and April 2013 was undertaken. Literature was screened and data were extracted by two independent reviewers. Studies were included of adult TIA/minor stroke participants with any of the outcomes of interest: fatigue, anxiety, depression, PTSD, cognitive impairment, QoL, change in emotions and return to work. Random-effects meta-analysis pooled outcomes by measurement tool. Searches identified 5976 records, 289 were assessed for eligibility and 31 studies were included. Results suggest high levels of cognitive impairment and depression post-TIA/minor stroke which decreased over time. However, frequencies varied between studies. Limited information was available on anxiety, PTSD and fatigue. Meta-analysis revealed that the measurement tool administered influenced the prevalence of cognitive impairment: Mini-Mental State Examination 17% [95% confidence interval (CI) 7, 26]; neuropsychological test battery 39% (95% CI 28, 50); Montreal Cognitive Assessment 54% (95% CI 43, 66). There is evidence to suggest that TIA/minor stroke patients may experience residual impairments; however, results should be interpreted with caution because of the few high quality studies. Notwithstanding, it is important to raise awareness of potential subtle but meaningful residual impairments.

Avapritinib-based SAR studies unveil a binding pocket in KIT and PDGFRA.

Avapritinib is the only potent and selective inhibitor approved for the treatment of D842V-mutant gastrointestinal stromal tumors (GIST), the most common primary mutation of the platelet-derived growth factor receptor α (PDGFRA). The approval was based on the NAVIGATOR trial, which revealed overall response rates of more than 90%. Despite this transformational activity, patients eventually progress, mostly due to acquired resistance mutations or following discontinuation due to neuro-cognitive side effects. These patients have no therapeutic alternative and face a dismal prognosis. Notable, little is known about this drug's binding mode and its medicinal chemistry development, which is instrumental for the development of the next generation of drugs. Against this background, we solve the crystal structures of avapritinib in complex with wild-type and mutant PDGFRA and stem cell factor receptor (KIT), which provide evidence and understanding of inhibitor binding and lead to the identification of a sub-pocket (Gα-pocket). We utilize this information to design, synthesize and characterize avapritinib derivatives for the determination of key pharmacophoric features to overcome drug resistance and limit potential blood-brain barrier penetration.

Molecular remodeling mechanisms of the neural somatodendritic compartment.

Neuronal cells use the process of vesicle trafficking to manipulate the populations of neurotransmitter receptors and other membrane proteins. Long term potentiation (LTP) is a long-lived increase in synaptic strength between neurons and increases postsynaptic dendritic spine size and the concentration of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate-type glutamate receptor (AMPAR) located in the postsynaptic density. AMPAR is removed from the cell surface via clathrin-mediated endocytosis. While the adaptor protein 2 (AP2) complex of endocytosis seems to have the components needed to allow temporal and spatial regulations of internalization, many accessory proteins are involved, such as epidermal growth factor receptor phosphorylation substrate 15 (Eps15). A sequence of repeats in the Eps15 protein is known as the Eps15 homology (EH) domain. It has affinity for asparagine-proline-phenylalanine (NPF) sequences that are contained within vesicle trafficking proteins such as epsin, Rab11 family interacting protein 2 (Rab11-FIP2), and Numb. After endocytosis, a pool of AMPAR is stored in the endosomal recycling compartment that can be transported to the dendritic spine surface upon stimulation during LTP for lateral diffusion into the postsynaptic density. Rab11 and the Eps15 homologue EHD1 are involved in receptor recycling. EHD family members are also involved in transcytosis of the neuronal cell adhesion molecule neuron-glia cell adhesion molecule (NgCAM) from the somatodendritic compartment to the axon. Neurons have a unique morphology comprising many projections of membrane that is constructed in part by the effects of the Eps15 homologue, intersectin. Morphogenesis in the somatodendritic compartment is becoming better understood, but there is still much exciting territory to explore, especially regarding the roles of various EH domain-NPF interactions in endocytic and recycling processes.

Population genetic structure of variable drug response.

Geographic patterns of genetic variation, including variation at drug metabolizing enzyme (DME) loci and drug targets, indicate that geographic structuring of inter-individual variation in drug response may occur frequently. This raises two questions: how to represent human population genetic structure in the evaluation of drug safety and efficacy, and how to relate this structure to drug response. We address these by (i) inferring the genetic structure present in a heterogeneous sample and (ii) comparing the distribution of DME variants across the inferred genetic clusters of individuals. We find that commonly used ethnic labels are both insufficient and inaccurate representations of the inferred genetic clusters, and that drug-metabolizing profiles, defined by the distribution of DME variants, differ significantly among the clusters. We note, however, that the complexity of human demographic history means that there is no obvious natural clustering scheme, nor an obvious appropriate degree of resolution. Our comparison of drug-metabolizing profiles across the inferred clusters establishes a framework for assessing the appropriate level of resolution in relating genetic structure to drug response.

Pathophysiology, staging and therapy of severe sepsis in baboon models.

We review our baboon models of Escherichia coli sepsis that mimic, respectively, the shock/disseminated intravascular coagulation (DIC) and organ failure variants of severe sepsis, and analyse the pathophysiologic processes that are unique to each. The multi-stage, multi-factorial characteristics of severe sepsis develop as a result of the initial insult, which - depending on its intensity - activates components of the intravascular compartment leading to overwhelming shock/DIC; or initiates a sequence of events involving both the intra- and extravascular (tissues) compartments that lead to organ failure. In the latter case, the disorder passes through two stages: an initial inflammatory/coagulopathic intravascular first stage triggered by E. coli, followed by an extravascular second stage, involving components unique to each organ and triggered by ischemia/reperfusion (oxidative stress and histone release). Although a myriad of overlapping cellular and molecular components are involved, it is the context in which these components are brought into play that determine whether shock/DIC or organ failure predominate. For example, inflammatory and thrombotic responses amplified by thrombin in the first case whereas similar responses are amplified by complement activation products in the second. Rather than blocking specific mediators, we found that attenuation of the thrombin and complement amplification pathways can effectively reverse the shock/DIC and organ failure exhibited by the LD(100) and LD(50) E. coli models of severe sepsis, respectively. Translation of these concepts to successful intervention in the respective baboon models of E. coli sepsis and the application to their clinical counterparts is described.

Complement inhibition decreases the procoagulant response and confers organ protection in a baboon model of Escherichia coli sepsis.

Severe sepsis leads to massive activation of coagulation and complement cascades that could contribute to multiple organ failure and death. To investigate the role of the complement and its crosstalk with the hemostatic system in the pathophysiology and therapeutics of sepsis, we have used a potent inhibitor (compstatin) administered early or late after Escherichia coli challenge in a baboon model of sepsis-induced multiple organ failure. Compstatin infusion inhibited sepsis-induced blood and tissue biomarkers of complement activation, reduced leucopenia and thrombocytopenia, and lowered the accumulation of macrophages and platelets in organs. Compstatin decreased the coagulopathic response by down-regulating tissue factor and PAI-1, diminished global blood coagulation markers (fibrinogen, fibrin-degradation products, APTT), and preserved the endothelial anticoagulant properties. Compstatin treatment also improved cardiac function and the biochemical markers of kidney and liver damage. Histologic analysis of vital organs collected from animals euthanized after 24 hours showed decreased microvascular thrombosis, improved vascular barrier function, and less leukocyte infiltration and cell death, all consistent with attenuated organ injury. We conclude that complement-coagulation interplay contributes to the progression of severe sepsis and blocking the harmful effects of complement activation products, especially during the organ failure stage of severe sepsis is a potentially important therapeutic strategy.

Thrombin-activatable fibrinolysis inhibitor is activated in vivo in a baboon model of Escherichia coli induced sepsis.

Activated thrombin-activatable fibrinolysis inhibitor (TAFIa or CPU) is a carboxypeptidase that is able to attenuate fibrinolysis. Although its role in fibrinolysis and inflammation has been studied extensively in vitro, its levels and subsequent effect in vivo has not been studied to the same extent. Using our recently developed assay that is specific for TAFIa, we were able to quantify its levels in plasma samples obtained from an Escherichia coli (E. coli) challenged baboon sepsis model. TAFIa levels accumulated appeared to be E. coli dose dependent, where the lethal dose of 10(10) CFU/kg generated a peak TAFIa level of 24 nM by 2 h, which represents almost 32% of total plasma level of its precursor, thrombin-activatable fibrinolysis inhibitor (TAFI or proCPU). Furthermore, our data suggest that there is continual TAFI activation under lethal level of E. coli as the apparent half-life of TAFIa is increased from 8 min to 2.2 h. Two sublethal doses of 10(8) and 10(6) CFU/kg generated peak TAFIa levels of 1.1 and 0.4 nM, respectively, both by 6 h. Taken together, our data show that TAFIa is generated at systemic levels, in a dose-dependent manner, that can substantially affect both fibrinolysis and inflammatory response in the E. coli challenged baboon sepsis model.

Perceptions and experiences of blood pressure self-monitoring during hypertensive pregnancy: A qualitative analysis of women's and clinicians' experiences in the OPTIMUM-BP trial.

BACKGROUND: Self-monitoring of blood pressure (BP) has been shown to be effective at improving BP control in the general population. The OPTIMUM-BP feasibility study was a prospective randomised controlled trial of self-monitoring of BP (SMBP) during hypertensive pregnancy. OBJECTIVE: To explore experiences, perceptions, and use of the OPTIMUM-BP self-monitoring intervention. STUDY DESIGN: Qualitative study within the OPTIMUM-BP feasibility trial. Semi-structured interviews with a purposive sample of pregnant women with chronic hypertension (n = 24) and their clinicians (n = 8) as well as 38 ethnographic observations of antenatal visits. RESULTS: Women found self-monitoring of BP feasible and acceptable and were highly motivated and pro-active in their monitoring, reporting greater control and knowledge of BP and reassurance. Women's persistence with SMBP was driven by a perceived need to safeguard the pregnancy, particularly among those taking antihypertensive medication. Clinicians also described the intervention as acceptable, though BP variability could cause uncertainty. Clinicians used different heuristics to integrate home and clinic readings. Observations suggested close working relationships between women and clinicians were key for confident integration of self-monitoring. CONCLUSIONS: Self-monitoring of BP was acceptable both to pregnant women with hypertension and their clinicians. More research is needed to understand BP variability within pregnancy to help interpret and integrate home BP readings for improved BP management. Clinical pathways that use BP self-monitoring should aim to maintain the continuity of care and relationships that are valued and appear pivotal for the confident and safe use of self-monitoring in pregnancy.

Bromocriptine in the treatment of alcoholics with the D2 dopamine receptor A1 allele.

Various types of alcoholics have been described and heredity has been shown to be involved in some of these types. An important role of the mesolimbic dopamine system has been suggested in the reinforcing effects of alcohol and recent molecular genetic studies are implicating the gene for the D2 dopamine receptor (DRD2) in alcoholism. In a double-blind study, bromocriptine, a DRD2 agonist, or placebo was administered to alcoholics with either the A1 (A1/A1 and A1/A2 genotypes) or only the A2 (A2/A2 genotype) allele of the DRD2 gene. The greatest improvement in craving and anxiety occurred in the bromocriptine-treated A1 alcoholics and attrition was highest in the placebo-treated A1 alcoholics. The feasibility of a pharmacogenetic approach in treating certain types of alcoholics is suggested.

Changes to management of hypertension in pregnancy, and attitudes to self-management: An online survey of obstetricians, before and following the first wave of the COVID-19 pandemic.

OBJECTIVE: This study aimed to understand the views and practice of obstetricians regarding self-monitoring for hypertensive disorders of pregnancy (blood pressure (BP) and proteinuria), the potential for self-management (including actions taken on self-monitored parameters) and to understand the impact of the COVID-19 pandemic on such views. DESIGN: Cross-sectional online survey pre- and post- the first wave of the COVID-19 pandemic. SETTING AND SAMPLE: UK obstetricians recruited via an online portal. METHODS: A survey undertaken in two rounds: December 2019-January 2020 (pre-pandemic), and September-November 2020 (during pandemic) RESULTS: 251 responses were received across rounds one (150) and two (101). Most obstetricians considered that self-monitoring of BP and home urinalysis had a role in guiding clinical decisions and this increased significantly following the first wave of the COVID-19 pandemic (88%, (132/150) 95%CI: 83-93% first round vs 96% (95%CI: 92-94%), (97/101), second round; p = 0.039). Following the pandemic, nearly half were agreeable to women self-managing their hypertension by using their own readings to make a pre-agreed medication change themselves (47%, 47/101 (95%CI: 37-57%)). CONCLUSIONS: A substantial majority of UK obstetricians considered that self-monitoring had a role in the management of pregnancy hypertension and this increased following the pandemic. Around half are now supportive of women having a wider role in self-management of hypertensive treatment. Maximising the potential of such changes in pregnancy hypertension management requires further work to understand how to fully integrate women's own measurements into clinical care.

Indoleamine 2,3-dioxygenase attenuates inhibitor development in gene-therapy-treated hemophilia A mice.

A serious impediment to gene and protein replacement therapy in hemophilia A is the development of inhibitors. Mechanisms responsible for inhibitor development include T-cell-dependent adaptive immune responses and the CD28-B7 signaling pathway that eventually leads to the formation of antibodies directed against factor VIII (FVIII). Indoleamine 2,3-dioxygenase (IDO) is a potent immunosuppressive enzyme that can inhibit T-cell responses and induce T-cell apoptosis by regulation of tryptophan metabolism. Kynurenine, one of the metabolites of tryptophan, has been implicated as an immune modulator. Here we hypothesize that co-delivery of the genes for FVIII and IDO can attenuate inhibitor formation. Using transposon-based gene delivery, we observed long-term therapeutic FVIII expression and significantly reduced inhibitor titers when the genes were co-delivered. Co-expression of FVIII and IDO in the liver was associated with increased plasma kynurenine levels, an inhibition of T-cell infiltration and increased apoptosis of T cells within the liver. These experiments suggest that modulation of tryptophan catabolism through IDO expression provides a novel strategy to reduce inhibitor development in hemophilia gene/protein therapy.

Peroxidation of subcellular organelles: formation of lipofuscinlike fluorescent pigments.

Lipid peroxidation of subcellular organelles gives fluorescent products which have fluorescence and excitation spectra similar to those of lipofuscin pigments. Fluorescence and excitation spectra and total fluorescence in the 460-nanometer region are useful for qualitative identification and quantitative measurement of the Schiff base product, a molecular damage site of lipid peroxidation which develops during some aging processes.

Dynamic optical lattices: two-dimensional rotating and accordion lattices for ultracold atoms.

We demonstrate a novel experimental arrangement which can rotate a 2D optical lattice at frequencies up to several kilohertz. Ultracold atoms in such a rotating lattice can be used for the direct quantum simulation of strongly correlated systems under large effective magnetic fields, allowing investigation of phenomena such as the fractional quantum Hall effect. Our arrangement also allows the periodicity of a 2D optical lattice to be varied dynamically, producing a 2D accordion lattice.

Nonnightmare distressed awakenings in veterans with posttraumatic stress disorder: response to prazosin.

Twenty-two veterans with posttraumatic stress disorder (PTSD) were assessed for trauma-related nightmares and nonnightmare distressed awakenings (NNDA) before and after treatment with the alpha-1 adrenoreceptor antagonist prazosin at an average bedtime dose of 9.6 mg/day. Ratings combining frequency and intensity dimensions of trauma-related nightmares decreased from 3.6 to 2.2, NNDA from 5.2 to 2.1, and sleep difficulty from 7.2 to 4.1 per week. These results suggest that increased brain adrenergic activity may contribute to the pathophysiology of both trauma-related nightmares and NNDA in PTSD.