The Joule heating problem in silver nanowire transparent electrodes.

Silver nanowire transparent electrodes have shown considerable potential to replace conventional transparent conductive materials. However, in this report we show that Joule heating is a unique and serious problem with these electrodes. When conducting current densities encountered in organic solar cells, the average surface temperature of indium tin oxide (ITO) and silver nanowire electrodes, both with sheet resistances of 60 ohms/square, remains below 35 °C. However, in contrast to ITO, the temperature in the nanowire electrode is very non-uniform, with some localized points reaching temperatures above 250 °C. These hotspots accelerate nanowire degradation, leading to electrode failure after 5 days of continuous current flow. We show that graphene, a commonly used passivation layer for these electrodes, slows nanowire degradation and creates a more uniform surface temperature under current flow. However, the graphene does not prevent Joule heating in the nanowires and local points of high temperature ultimately shift the failure mechanism from nanowire degradation to melting of the underlying plastic substrate. In this paper, surface temperature mapping, lifetime testing under current flow, post-mortem analysis, and modelling illuminate the behaviour and failure mechanisms of nanowires under extended current flow and provide guidelines for managing Joule heating.

Impulsivity in borderline personality disorder.

BACKGROUND: Impulsivity is a core feature of borderline personality disorder (BPD) and is most frequently measured using self-rating scales. There is a need to find objective, valid and reliable measures of impulsivity. This study aimed to examine performance of participants with BPD compared with healthy controls on delay and probabilistic discounting tasks and the stop-signal task (SST), which are objective measures of choice and motor impulsivity, respectively. METHOD: A total of 20 participants with BPD and 21 healthy control participants completed delay and probabilistic discounting tasks and the SST. They also completed the Barratt Impulsiveness Scale (BIS), a self-rating measure of impulsivity. RESULTS: Participants with BPD showed significantly greater delay discounting than controls, manifest as a greater tendency to accept the immediately available lesser reward rather than waiting longer for a greater reward. Similarly they showed significantly greater discounting of rewards by the probability of payout, which correlated with past childhood trauma. Participants with BPD were found to choose the more certain and/or immediate rewards, irrespective of the value. On the SST the BPD and control groups did not differ significantly, demonstrating no difference in motor impulsivity. There was no significant difference between groups on self-reported impulsivity as measured by the BIS. CONCLUSIONS: Measures of impulsivity show that while motor impulsivity was not significantly different in participants with BPD compared with controls, choice or reward-related impulsivity was significantly affected in those with BPD. This suggests that choice impulsivity but not motor impulsivity is a core feature of BPD.

A feasibility study testing four hypotheses with phase II outcomes in advanced colorectal cancer (MRC FOCUS3): a model for randomised controlled trials in the era of personalised medicine?

BACKGROUND: Molecular characteristics of cancer vary between individuals. In future, most trials will require assessment of biomarkers to allocate patients into enriched populations in which targeted therapies are more likely to be effective. The MRC FOCUS3 trial is a feasibility study to assess key elements in the planning of such studies. PATIENTS AND METHODS: Patients with advanced colorectal cancer were registered from 24 centres between February 2010 and April 2011. With their consent, patients' tumour samples were analysed for KRAS/BRAF oncogene mutation status and topoisomerase 1 (topo-1) immunohistochemistry. Patients were then classified into one of four molecular strata; within each strata patients were randomised to one of two hypothesis-driven experimental therapies or a common control arm (FOLFIRI chemotherapy). A 4-stage suite of patient information sheets (PISs) was developed to avoid patient overload. RESULTS: A total of 332 patients were registered, 244 randomised. Among randomised patients, biomarker results were provided within 10 working days (w.d.) in 71%, 15 w.d. in 91% and 20 w.d. in 99%. DNA mutation analysis was 100% concordant between two laboratories. Over 90% of participants reported excellent understanding of all aspects of the trial. In this randomised phase II setting, omission of irinotecan in the low topo-1 group was associated with increased response rate and addition of cetuximab in the KRAS, BRAF wild-type cohort was associated with longer progression-free survival. CONCLUSIONS: Patient samples can be collected and analysed within workable time frames and with reproducible mutation results. Complex multi-arm designs are acceptable to patients with good PIS. Randomisation within each cohort provides outcome data that can inform clinical practice.

Suboptimal maternal nutrition during early fetal kidney development specifically promotes renal lipid accumulation following juvenile obesity in the offspring.

Reduced maternal food intake between early-to-mid gestation results in tissue-specific adaptations in the offspring following juvenile-onset obesity that are indicative of insulin resistance. The aim of the present study was to establish the extent to which renal ectopic lipid accumulation, as opposed to other markers of renal stress, such as iron deposition and apoptosis, is enhanced in obese offspring born to mothers nutrient restricted (NR) throughout early fetal kidney development. Pregnant sheep were fed either 100% (control) or NR (i.e. fed 50% of their total metabolisable energy requirement from 30-80 days gestation and 100% at all other times). At weaning, offspring were made obese and, at approximately 1 year, kidneys were sampled. Triglyceride content, HIF-1α gene expression and the protein abundance of the outer-membrane transporter voltage-dependent anion-selective channel protein (VDAC)-I on the kidney cortex were increased in obese offspring born to NR mothers compared with those born to controls, which exhibited increased iron accumulation within the tubular epithelial cells and increased gene expression of the death receptor Fas. In conclusion, suboptimal maternal nutrition coincident with early fetal kidney development results in enhanced renal lipid deposition following juvenile obesity and could accelerate the onset of the adverse metabolic, rather than cardiovascular, symptoms accompanying the metabolic syndrome.

Adipose tissue and fetal programming.

Adipose tissue function changes with development. In the newborn, brown adipose tissue (BAT) is essential for ensuring effective adaptation to the extrauterine environment, and its growth during gestation is largely dependent on glucose supply from the mother to the fetus. The amount, location and type of adipose tissue deposited can also determine fetal glucose homeostasis. Adipose tissue first appears at around mid-gestation. Total adipose mass then increases through late gestation, when it comprises a mixture of white and brown adipocytes. BAT possesses a unique uncoupling protein, UCP1, which is responsible for the rapid generation of large amounts of heat at birth. Then, during postnatal life some, but not all, depots are replaced by white fat. This process can be utilised to investigate the physiological conversion of brown to white fat, and how it is re-programmed by nutritional changes in pre- and postnatal environments. A reduction in early BAT deposition may perpetuate through the life cycle, thereby suppressing energy expenditure and ultimately promoting obesity. Normal fat development profiles in the offspring are modified by changes in maternal diet at defined stages of pregnancy, ultimately leading to adverse long-term outcomes. For example, excess macrophage accumulation and the onset of insulin resistance occur in an adipose tissue depot-specific manner in offspring born to mothers fed a suboptimal diet from early to mid-gestation. In conclusion, the growth of the different fetal adipose tissue depots varies according to maternal diet and, if challenged in later life, this can contribute to insulin resistance and impaired glucose homeostasis.

Effect of two sitting postures on lumbar sagittal alignment and intervertebral discs in runners.

PURPOSE: We evaluated in vivo changes in lumbar lordosis and intervertebral discs in runners and assessed the relationship between these changes and degenerative disc disease in runners with and without a history of low back pain. MATERIALS AND METHODS: Using open upright magnetic resonance (MR) imaging, we prospectively studied changes in lumbar lordosis and intervertebral discs of 25 elite long-distance runners in two sitting postures (neutral and extended) before and after 1 h of running and compared the results with disc height and dehydration/degeneration. Seventeen of the 25 runners had a history of low back pain. RESULTS: After 1 h of running, mean lordosis in neutral posture reduced by 4°; reduction was significant in runners with a history of low back pain. A significant reduction in mean lordosis in extension was not observed. Mean disc height significantly reduced in both postures, without, however, any statistical significance between runners with and without a history low back pain in any posture. Variable degrees of disc dehydration/degeneration were observed in 23 runners (57 discs), more commonly at L5-S1. A significant difference of disc dehydration/degeneration between runners with and without a history of low back pain was not observed. CONCLUSIONS: Intervertebral discs undergo significant strain after 1 h of running that in the long term may lead to low back pain and degenerative disc disease. Runners, especially those with low back pain and degenerative disc disease, should be evaluated after training to preserve the normal lumbar lordosis.

Emerging dominance of summer rainfall driving High Arctic terrestrial-aquatic connectivity.

Hydrological transformations induced by climate warming are causing Arctic annual fluvial energy to shift from skewed (snowmelt-dominated) to multimodal (snowmelt- and rainfall-dominated) distributions. We integrated decade-long hydrometeorological and biogeochemical data from the High Arctic to show that shifts in the timing and magnitude of annual discharge patterns and stream power budgets are causing Arctic material transfer regimes to undergo fundamental changes. Increased late summer rainfall enhanced terrestrial-aquatic connectivity for dissolved and particulate material fluxes. Permafrost disturbances (<3% of the watersheds' areal extent) reduced watershed-scale dissolved organic carbon export, offsetting concurrent increased export in undisturbed watersheds. To overcome the watersheds' buffering capacity for transferring particulate material (30 ± 9 Watt), rainfall events had to increase by an order of magnitude, indicating the landscape is primed for accelerated geomorphological change when future rainfall magnitudes and consequent pluvial responses exceed the current buffering capacity of the terrestrial-aquatic continuum.

Low levels of HIV-1 infection in cutaneous dendritic cells promote extensive viral replication upon binding to memory CD4+ T cells.

Earlier work has identified a cell population that replicates HIV-1 in the absence of standard T cell stimuli. The system consists of dendritic cells and memory T lymphocytes that emigrate from organ cultures of human skin and together support a productive infection with HIV-1. These emigrants resemble cells that can be found in mucous membranes and that normally traffic in afferent lymph. Here, we report that a low level of infection in the dendritic cell can initiate extensive HIV-1 replication in cocultures with T cells. First we extended our earlier work to larger skin specimens from cadavers. As long as the organ cultures were set up within 36 h of death, the emigrant leukocytes were comparable to cells from fresh surgical specimens in number, phenotype, and function. These mixtures of dendritic cells and T cells provided the milieu for a productive infection with several virus isolates. When purified dendritic cells were separately pulsed with virus and then mixed with T cells that had not been pulsed with HIV-1, active infection ensued. The infectivity of HIV-pulsed dendritic cells persisted for at least 1.5 d in culture, but was blocked if AZT was added during that time to block reverse transcription in the dendritic cells. The number of copies of proviral DNA in the dendritic cells corresponded to < 100 copies per 5 X 10(4) cells, but upon mixing with T cells, > 10(4) copies were found 5-7 d later. By contacting syngeneic T cells, extralymphoid depots of dendritic cells--even with a low viral burden as has been reported in vivo--may contribute to chronic HIV-1 replication in infected individuals.

Dendritic cells freshly isolated from human blood express CD4 and mature into typical immunostimulatory dendritic cells after culture in monocyte-conditioned medium.

A procedure has been developed to isolate dendritic cells to a high degree of purity from fresh blood. Prior enrichment methods have relied upon an initial 1-2-d culture period. Purified fresh isolates lack the characteristic morphology, phenotype, and immunostimulatory function of dendritic cells. The purified cells have the appearance of medium sized lymphocytes and express substantial levels of CD4, but lack the T cell molecules CD3, CD8, and T cell receptor. When placed in culture, the cells mature in a manner resembling the previously described, cytokine-dependent maturation of epidermal dendritic cells (Langerhans cells). The cells enlarge and exhibit many cell processes, express much higher levels of major histocompatibility complex class II and a panel of accessory molecules for T cell activation, and become potent stimulators of the mixed leukocyte reaction. Among the many changes during this maturation process are a fall in CD4 and the appearance of high levels of B7/BB1, the costimulator for enhanced interleukin 2 production in T cells. These changes are not associated with cell proliferation, but are dependent upon the addition of monocyte-conditioned medium. We suggest that the freshly isolated CD4-positive blood dendritic cells are recent migrants from the bone marrow, and that subsequent maturation of the lineage occurs in tissues in situ upon appropriate exposure to cytokines.

Efficient interaction of HIV-1 with purified dendritic cells via multiple chemokine coreceptors.

HIV-1 actively replicates in dendritic cell (DC)-T cell cocultures, but it has been difficult to demonstrate substantial infection of purified mature DCs. We now find that HIV-1 begins reverse transcription much more efficiently in DCs than T cells, even though T cells have higher levels of CD4 and gp120 binding. DCs isolated from skin or from blood precursors behave similarly. Several M-tropic strains and the T-tropic strain IIIB enter DCs efficiently, as assessed by the progressive formation of the early products of reverse transcription after a 90-min virus pulse at 37 degrees C. However, few late gag-containing sequences are detected, so that active viral replication does not occur. The formation of these early transcripts seems to follow entry of HIV-1, rather than binding of virions that contain viral DNA. Early transcripts are scarce if DCs are exposed to virus on ice for 4 h, or for 90 min at 37 degrees C, conditions which allow virus binding. Also the early transcripts once formed are insensitive to trypsin. The entry of a M-tropic isolates is blocked by the chemokine RANTES, and the entry of IIIB by SDF-1. RANTES interacts with CCR5 and SDF-1 with CXCR4 receptors. Entry of M-tropic but not T-tropic virus is ablated in DCs from individuals who lack a functional CCR5 receptor. DCs express more CCR5 and CXCR4 mRNA than T cells. Therefore, while HIV-1 does not replicate efficiently in mature DCs, viral entry can be active and can be blocked by chemokines that act on known receptors for M- and T-tropic virus.

Accuracy of polar F6 in estimating the energy cost of aerobic dance bench stepping in college-age females.

AIM: This study: 1) examined the accuracy of the Polar F6 for estimating energy expenditure (EE) in a sample of college-age women during aerobic dance bench stepping (ADBS) using predicted maximal oxygen consumption (VO2max) and maximal heart rate (HRmax), and 2) determined whether the use of actual measures of VO2max and HRmax improves the accuracy of the Polar F6 for estimating EE. METHODS: Thirty-two females had their VO2max and HRmax predicted by the Polar F6 heart rate monitor (HRM), and then performed a graded maximal exercise treadmill test to determine their actual VO2max and HRmax. The participants then followed a 20-min ADBS routine while stepping up and down off of a 15.24-cm bench at a cadence of 126 beats.min-1. During ADBS, the participants wore two F6 HRM that simultaneously collected data. To estimate EE, one HRM utilized their predicted VO2max and HRmax (PHRM) while the other HRM utilized their actual VO2max and HRmax (AHRM). RESULTS: The predicted HRmax significantly overestimated actual HRmax by 3.75 beats.min-1 on average, and the predicted VO2max overestimated actual VO2max by 2.63 ml.kg-1.min-1 on average (P<0.01). However, there were no significant differences between the PHRM and AHRM (P≥0.05). When compared to indirect calorimetry, the PHRM and AHRM significantly overestimated average EE by 28% (2.4 kcal.min-1) and 27% (2.0 kcal.min-1), respectively (P<0.05). CONCLUSIONS: Even when using actual measures of VO2max and HRmax, the Polar F6 is inaccurate in estimating EE during ADBS for college-age females.

Safety of Consecutive Bilateral Decubitus Digital Subtraction Myelography in Patients with Spontaneous Intracranial Hypotension and Occult CSF Leak.

BACKGROUND AND PURPOSE: Digital subtraction myelography performed with the patient in the lateral decubitus position has the potential for increased sensitivity over prone-position myelography in the detection of spinal CSF-venous fistulas, a well-established cause of spontaneous intracranial hypotension. We report on the safety of performing routine, consecutive-day right and left lateral decubitus digital subtraction myelography in these patients. MATERIALS AND METHODS: In this retrospective case series, all patients undergoing consecutive-day lateral decubitus digital subtraction myelography for suspected spinal CSF leak between September 2018 and September 2019 were identified. Chart review was performed to identify any immediate or delayed adverse effects associated with the procedures. Procedural parameters were also analyzed due to inherent variations associated with the positive-pressure myelography technique that was used. RESULTS: A total of 60 patients underwent 68 pairs of consecutive-day lateral decubitus digital subtraction myelographic examinations during the study period. No major adverse effects were recorded. Various minor adverse effects were observed, including pain requiring analgesics (27.2%), nausea/vomiting requiring antiemetics (8.1%), and transient neurologic effects such as syncope, vertigo, altered mental status, and autonomic dysfunction (5.1%). Minor transient neurologic effects were correlated with increasing volumes of intrathecal saline injectate used for thecal sac prepressurization. CONCLUSIONS: In patients with spontaneous intracranial hypotension and suspected spontaneous spinal CSF leak, consecutive-day lateral decubitus digital subtraction myelography demonstrates an acceptable risk profile without evidence of neurotoxic effects from cumulative intrathecal contrast doses. Higher intrathecal saline injectate volumes may correlate with an increased incidence of minor transient periprocedural neurologic effects.

Differential impact of thermal and physical permafrost disturbances on High Arctic dissolved and particulate fluvial fluxes.

Climate warming and changing precipitation patterns have thermally (active layer deepening) and physically (permafrost-thaw related mass movements) disturbed permafrost-underlain watersheds across much of the Arctic, increasing the transfer of dissolved and particulate material from terrestrial to aquatic ecosystems. We examined the multiyear (2006-2017) impact of thermal and physical permafrost disturbances on all of the major components of fluvial flux. Thermal disturbances increased the flux of dissolved organic carbon (DOC), but localized physical disturbances decreased multiyear DOC flux. Physical disturbances increased major ion and suspended sediment flux, which remained elevated a decade after disturbance, and changed carbon export from a DOC to a particulate organic carbon (POC) dominated system. As the magnitude and frequency of physical permafrost disturbance intensifies in response to Arctic climate change, disturbances will become an increasingly important mechanism to deliver POC from terrestrial to aquatic ecosystems. Although nival runoff remained the primary hydrological driver, the importance of pluvial runoff as driver of fluvial flux increased following both thermal and physical permafrost disturbance. We conclude the transition from a nival-dominated fluvial regime to a regime where rainfall runoff is proportionately more important will be a likely tipping point to accelerated High Arctic change.

Replication of HIV-1 in dendritic cell-derived syncytia at the mucosal surface of the adenoid.

Human immunodeficiency virus-type 1 (HIV-1) replicates actively in infected individuals, yet cells with intracellular depots of viral protein are observed only infrequently. Many cells expressing the HIV-1 Gag protein were detected at the surface of the nasopharyngeal tonsil or adenoid. This infected mucosal surface contained T cells and dendritic cells, two cell types that together support HIV-1 replication in culture. The infected cells were multinucleated syncytia and expressed the S100 and p55 dendritic cell markers. Eleven of the 13 specimens analyzed were from donors who did not have symptoms of acquired immunodeficiency syndrome (AIDS). The interaction of dendritic cells and T cells in mucosa may support HIV-1 replication, even in subclinical stages of infection.

Rapid infection of oral mucosal-associated lymphoid tissue with simian immunodeficiency virus.

The early events during infection with an immunodeficiency virus were followed by application of pathogenic simian immunodeficiency virus atraumatically to the tonsils of macaques. Analyses by virologic assays and in situ hybridization revealed that the infection started locally in the tonsils, a mucosal-associated lymphoid organ, and quickly spread to other lymphoid tissues. At day 3, there were few infected cells, but then the number increased rapidly, reaching a high plateau between days 4 and 7. The infection was not detected in the dendritic cell-rich squamous epithelium to which the virus was applied; instead, it was primarily in CD4+ tonsillar T cells, close to the specialized antigen-transporting epithelium of the tonsillar crypts. Transport of the virus and immune-activating stimuli across this epithelium would allow mucosal lymphoid tissue to function in the atraumatic transmission of immunodeficiency viruses.

Follicle and oocyte morphology in ewes after treatment with insulin in the late follicular phase.

Stress reduces fertility in ruminants. Various experimental models, such as insulin-induced hypoglycaemia, have been used to investigate the mechanisms involved, and have revealed abnormal LH profiles (both pulse and surge secretion). This disruption affects follicular function and it is proposed there may be negative consequences on subsequent oocyte morphology. Insulin (5iu/kg), administered to ewes in the late follicular phase, induced hypoglycemia for 10h, decreased estradiol concentrations for 8-12h and delayed the LH surge by 15h. Although the diameters of dominant follicles just before ovulation were not affected, granulosa cells had fewer pycnotic nuclei, less apoptosis and increased proliferation 16-17h after the LH surge. Nevertheless, we did not observe gross ultra-structural differences in nuclear, cytoplasmic or cumulus maturity between oocytes from insulin-treated and control animals. This suggests that reduced LH pulsatility and a delay in the LH surge may only produce very subtle changes in gross oocyte morphology, imperceptible by electron microscopy.

Effects of whole body vibration on spinal proprioception in healthy individuals.

BACKGROUND: Low back pain (LBP) is a common health problem with high reoccurrence rate. As patients with LBP are often found to be proprioception impaired, new proprioception exercises should be explored. Whole body vibration (WBV) has been proven to improve muscle function and proprioception. OBJECTIVE: The aim of this study was to determine the effects of WBV on spinal proprioception when WBV was administered in standing and seated postures. METHODS: Twenty healthy male individuals (mean age: 23.2±1.2 years) were recruited and randomly assigned to two WBV groups: WBV in standing or WBV in seated posture. Their body posture, lumbar repositioning ability, maximum reaching distance and lumbopelvic coordination during dynamic motion in flexion and extension were assessed before, immediately after, 30 minutes after and 1 hour after 5 minutes of WBV (18 Hz, 6 mm amplitude) exposure. A Mixed ANOVA was used to analyze the effects of group and time factors on these four outcome measures. RESULTS: There were no significant interaction (group and time) and group effects on all outcome measures. Participants were found to have significant different time effect on body posture, lumbar repositioning ability, maximum reaching distance and lumbopelvic coordination. CONCLUSIONS: WBV could significantly improve spinal proprioception including body posture, lumbar repositioning ability, maximum reaching distance and lumbopelvic coordination in healthy individuals. WBV protocol is recommended to confirm its clinical application for improving spinal proprioception and its effects on patients with LBP is warranted.

CpG-C ISS-ODN activation of blood-derived B cells from healthy and chronic immunodeficiency virus-infected macaques.

Cytosine-phosphate-guanine class C (CpG-C) immunostimulatory sequence oligodeoxynucleotides (ISS-ODNs) activate human B cells and dendritic cells (DCs), properties that suggest potential use as a novel adjuvant to enhance vaccine efficacy. After demonstrating that the CpG-C ISS-ODN C274 activates macaque DCs, we examined in vitro activation of macaque B cells by C274 as a prelude to evaluation of this molecule as an adjuvant in the testing of candidate human immunodeficiency virus vaccines in the rhesus macaque-simian immunodeficiency virus (SIV) model. C274 induced macaque CD20(+) B cells to proliferate more strongly than CD40 ligand or CpG-B ISS-ODN. C274 enhanced B cell survival; increased viability was most evident after 3-7 days of culture. Increased expression of CD40, CD80, and CD86 by B cells was apparent within 24 h of exposure to C274 and persisted for up to 1 week. C274-stimulated, B cell-enriched and peripheral blood mononuclear cell suspensions from naïve and immunodeficiency virus-infected monkeys secreted several cytokines [e.g., interleukin (IL)-3, IL-6, IL-12, interferon-alpha] and chemokines [e.g., monocyte chemoattractant protein-1/CC chemokine ligand 2 (CCL2), macrophage-inflammatory protein-1alpha/CCL3, IL-8/CXC chemokine ligand 8]. In comparison, exposure of macaque B cells to SIV had minimal impact on surface phenotype, despite inducing cytokine and chemokine production in cells from infected and uninfected animals. These observations emphasize the need to identify strategies to optimally boost immune function, as immunodeficiency viruses themselves only partially activate B cells and DCs. The ability of C274 to stimulate B cells and DCs in healthy and infected monkeys suggests its possible use as a broad-acting adjuvant to be applied in the rhesus macaque model for the development of preventative and therapeutic vaccines.

Rapid Proteomics to Prospect and Validate Novel Bacterial Metabolism Induced by Environmental Burden.

Understanding the pathophysiology of genes and enzymes involved in caffeine metabolism can have extracurricular benefits, such as providing distinct methylxanthines as intermediates for pharmaceutical synthesis, and also improve environmental waste remediation. The strains Pseudomonas putida CBB5 and CES may provide insights into these applications because they may both be induced to degrade caffeine, yet the latter thrives in concentrations >8.0gL-1; threefold higher than any other bacteria. We took a novel approach toward identifying the enzymatic pathways in both Pseudomonas sp. CES and a deletion mutation of strain CBB5, which largely circumvented the need for exhaustive isolation of enzymes and the stepwise reconstitution of their activities to determine caffeine response elements. Here, we describe two optimized, rapid alternative strategies based on multiplexed SIL assays and demonstrate their application by discovering caffeine-degrading enzymes in the CES strain based on quantitative comparison between enriched lysate fractions drawn from bacterial proteomes grown in the absence and presence of caffeine. Comparisons were made using stable isotope dimethyl labeling and expression differences were substantiated by reciprocal labeling experiments. The role of the identified proteins in caffeine degradation was independently verified by genetic sequencing. Multiple new components of N-demethylase system were discovered within a fraction of the lysate enriched specifically for this activity. We also describe how to expand the biological context (and reduce systemic bias) by adapting the protocol for total lysate analysis. We combined off-line prefractionation with the speed and resolution advantages of the Orbitrap LUMOS. The global protocol revealed 2406 proteins 1789 of which were quantified between treatments revealing, among other insights, a new antagonistic degradation pathway for vanillin that is completely suppressed by caffeine treatment.