Lithium is an anti-psychotic that has been shown to prevent the hyperphosphorylation of tau protein through the inhibition of glycogen-synthase kinase 3-beta (GSK3β). We recently developed a mouse model that progresses from amyloid pathology to tau pathology and neurodegeneration due to the genetic deletion of NOS2 in an APP transgenic mouse; the APPSwDI/NOS2−/− mouse. Because this mouse develops tau pathology, amyloid pathology and neuronal loss we were interested in the effect anti-tau therapy would have on amyloid pathology, learning and memory. We administered lithium in the diets of APPSwDI/NOS2−/− mice for a period of eight months, followed by water maze testing at 12 months of age, immediately prior to sacrifice. We found that lithium significantly lowered hyperphosphorylated tau levels as measured by Western blot and immunocytochemistry. However, we found no apparent neuroprotection, no effect on spatial memory deficits and an increase in histological amyloid deposition. Aβ levels measured biochemically were unaltered. We also found that lithium significantly altered the neuroinflammatory phenotype of the brain, resulting in enhanced alternative inflammatory response while concurrently lowering the classical inflammatory response. Our data suggest that lithium may be beneficial for the treatment of tauopathies but may not be beneficial for the treatment of Alzheimer's disease. Alzheimer's disease (AD) is a progressive, neurodegenerative disorder characterized clinically by an advancing cognitive decline. Pathologically, AD is identified by the presence of extracellular amyloid plaques composed of aggregated Aß peptides and intracellular neurofibrillary tangles (NFTs) composed of hyperphosphorylated, aggregated tau protein Tau protein is a microtubule associated protein that is involved in the stabilization of microtubules in a phosphorylation dependent manner Lithium is an antipsychotic medication that has been shown to inhibit glycogen-synthase kinase 3-beta (GSK3β); a key kinase for the phosphorylation of tau Radial-arm water maze testing showed that non-transgenic, wildtype (WT) and NOS2−/− mice aged 12 months learned the task as is evidenced by the decline in number of errors over the two days of testing, ending with a mean of less than 1 error at the end of day 2 . ImportaSince lithium has been associated with reduced tau hyperphosphorylation and subsequent aggregation we assessed tau phosphorylation by semi-quantitative Western blot. We observed a reduced signal for both AT8 (which recognizes tau phosphorylation at Ser202/Thr205) and AT180 (which recognizes tau phosphorylation at Thr231) . Both siTo determine the effects of lithium on Aβ, we assessed Aβ by both biochemical and immunohistochemical methods. As can be seen in We have previously shown that the APPSwDI/NOS2−/− mouse develops significant neuronal loss by 12 months of age To determine whether lithium had an influence on the microglial population we performed immunocytochemistry for CD11b and CD45. CD11b is a standard marker for all states of microglial cells that increases in intensity with activation To further characterize the inflammatory changes that might occur as a result of the lithium treatment we performed quantitative real-time RT-PCR for genes associated with classical and alternative inflammatory responses. We have previously shown that transgenic mice show diverse inflammatory responses and anti-Aβ immunotherapy significantly alters these responses The effect of lithium to significantly lower hyperphosphorylation of tau protein in mouse models of tauopathies was clearly shown by Noble et al We recently developed a mouse model of Alzheimer's disease that develops amyloid pathology as well as endogenous mouse tau pathology and significant neurodegeneration GSK3β is a serine/threonine protein kinase that has been shown to be involved in the phosphorylation of tau protein. In particular, GSK3β has been linked to the abnormal phosphorylation of tau in AD, and other tauopathies Despite these beneficial effects of lithium treatment on tau, lithium did not alter total brain levels of soluble and insoluble Aβ38, 40 and 42 and did not protect the neurons from degeneration. Thus, the tau-lowering action of lithium appears to be independent of Abeta and is unlikely to have altered the production or clearance kinetics of the Aβ peptides. Interestingly, the effect of lithium to increase observable amyloid deposition in the brain suggests that lithium may directly or indirectly alter the environment of the brain to promote the deposition of Aβ.Neuroinflammation is known to be present in the AD brain and has been hypothesized to be intimately involved in the control of AD pathologies Lithium is known to influence inflammation. Rapaport and Manji showed that lithium results in an increase in Th2 cytokines IL-4 and IL-10 along with a decrease in the Th1 cytokines IFNγ and IL-2 in an ex vivo assay on whole blood cultures In summary, we find that lithium treatment of the APPSwDI/NOS2−/− mouse results in decreased tau hyperphosphorylation, increased amyloid deposition, altered neuroinflammation and no change in neurodegeneration or memory.The study was approved by the Duke University Institutional Animal Care and Use Committee and conformed to the National Institutes of Health Guide for the Care and Use of Animals in Research. The APPSwDI/NOS2−/− mice (produced by crossing APPSwDI mice Mice (12 months old) were tested for memory and learning two days prior to sacrifice using the two-day radial-arm water maze as described in detail previously After injection with a lethal dose of ketamine the mice were perfused intracardially with 25 ml normal saline. Brains were rapidly removed and bisected in the mid-sagittal plane. The left half was immersion fixed in 4% paraformaldehyde, while the right half was snap-frozen in liquid nitrogen and stored at −80°C. The left hemibrain was passed through a series of 10, 20 and 30% sucrose solutions as cryoprotection and 25 µm frozen horizontal sections were collected using a sliding microtome and a freezing stage as described previously Eight 25 µm sections equally spaced 600 mm apart were selected for free floating immunohistochemistry for Aβ , neuN , PHF-tau , CD45  and CD11b . The method for free-floating immunohistochemistry has been described previously Immunohistochemical product was quantified by assessing percent area occupied by positive stain using the Nikon Elements BR software package . Briefly, images were collected on a Nikon Eclipse 90i upright microscope equipped with a Nikon DS-Ri1 digital camera. Specific landmarks on the tissue section were used to ensure the correct regions were examined. Fields of the frontal cortex and hippocampus were localized using 100× magnification followed by image collection at 200× magnification. Representative images were used to establish thresholds using Hue, Saturation and Intensity (HSI) values. The threshold file was saved and then applied to all images from all samples of a given immunostain to yield individual percent area occupied values for each image. Approximately six images of frontal cortex and four images of the hippocampus were assessed for each animal.(−delta delta Ct) method RNA was extracted from approximately 40 mg frozen pulverized tissue using the RNeasy tissue kit  according to the manufacturer's instructions. RNA was quantified using the nanodrop spectrophotometer  and cDNA produced using the cDNA High Capacity kit  according to the manufacturer's instructions. Real-time PCR was performed using the TaqMan Gene Expression assay kit  according to the manufacturer's instructions and as previously described Approximately 60 mg of the brain powder was homogenized and protein lysates were prepared in M-per lysis buffer  containing 1% complete protease/phosphatase inhibitor . Protein concentrations were assessed using the BCA protein assay kit , according to manufacturer's instructions. 15 µg protein from each lysate was run on a denaturing 4–20% SDS-PAGE gel. The gel was transferred onto a PVDF membrane using the iBlot system , and Western blots were performed for PFH Tau AT8  and AT180 . The blots were stripped using 5× New Blot Nitro Stripping Buffer  and re-probed using the above protocol for with ß-actin as loading control. Semi-quantitative densitometry analysis was performed using the Odyssey Imaging Software . Individual densitometry values were normalized to the β-actin densitometry value on the same blot.For ELISA measurement of Aβ we performed a two step protein extraction. 150 mg brain powder was first extracted in 250 µl PBS containing 1% complete protease/phosphatase inhibitor . The homogenate was centrifuged at 16,000×g at 4°C for 30 minutes. The supernatant was removed and became the “soluble” extract. The resulting pellet was then homogenized in 100 µl 70% formic acid and centrifuged at 16,000×g at 4°C for 30 minutes. The supernatant was removed, neutralized 1∶20 with 1 M Tris-HCl and became the “insoluble” extract. Protein concentration for both the soluble and insoluble extracts was determined using the BCA protein assay according to manufacturer's instructions. We used the Meso-Scale Discovery multiplex ELISA system to measure soluble and insoluble Aβ38, Aβ40 and Aβ42 . ELISA kits were run according to the manufacturer's instructions.Neurons that were positive for cresyl violet were counted in the cornu ammonis 3 (CA3) and the subiculum using the optical fractionator method of stereological counting  and the Olympus CAST 1 stereology system  connected to an upright Olympus microscope. The regions of interests (ROI) were defined using specific landmarks within the hippocampus to maintain consistency. A grid was placed randomly over the region of interest slated for counting. At regularly predetermined positions of the grid, cells were counted within three-dimensional optical disectors. Within each dissector, a 1 µm guard distance from the top and bottom of the section surface was excluded. Section thickness was measured regularly on all collected sections to estimate the mean section thickness for each animal after tissue processing and averaged 14.64 µm±0.29 µm for all sections analyzed. The total number of neurons was calculated using the equation:t test or two-way ANOVA. All immunohistochemical, stereological, ELISA, Western blot and qRT-PCR data were analyzed by one-way ANOVA. The statistical analysis software JMP  was used for all statistical analyses with p<0.05 judged as significant. All graphs were made using Graphpad Prism 4 .The significance of genotype- and treatment-specific behavioral changes were analyzed by the unpaired Student's


The concept of frustrated Lewis pairs (FLPs) has received considerable attention of late, and numerous reports have demonstrated the power of non- or weakly interacting Lewis acid–base pairs for the cooperative activation of small molecules. Although most studies have focused on the use of organic or main-group FLPs that utilize steric encumbrance to prevent adduct formation, a related strategy can be envisioned for both organic and inorganic complexes, in which "electronic frustration" engenders reactivity consistent with both nucleophilic (basic) and electrophilic (acidic) character. Here we propose that such a description is consistent with the behavior of many coordinatively unsaturated transition-metal species featuring metal–ligand multiple bonds, and we further demonstrate that the resultant reactivity may be a powerful tool for the functionalization of C–H and E–H bonds. As with the Schrock neopentylidene, the reaction proceeds through a four-membered metallacycle, which eliminates the organic product through a [2 + 2] cycloreversion  28]. Ot. Ot28]. The reactions described above represent only a few of the many metal–ligand cooperative reactions of nucleophilic, multiply bonded species with polar multiple bonds. Related reactions have been observed for terminal oxo, sulfido, phosphinidene, and alkylidyne complexes of early transition metals  and related variants such as alkyne and enyne metathesis  fashion across the Zr═NR bond . The result is a 1,2-addition of X–H across the M═E bond to give a M(X)(EH) species, which may in some cases react further.M2,in which the substrate is polarized in the presence of the frustrated pair and ultimately added across it . Th. Th42]. cross it . Intramocross it . Severalcross it –46, and δ+═Eδ− FLPs to activate some of the most challenging substrates.The limitations of these sorts of reactions in terms of potential catalytic applications are largely related to the reluctance of the metal center to undergo redox chemistry . The systems are constructed to favor high oxidation states, so reductive elimination is quite unfavorable relative to other non-redox processes, and insertion of unsaturated bonds is generally not observed. In a sense, this limitation is similar to what is encountered in attempts to use σ-bond metathesis processes in catalysis, in which only a few specialized systems have been reported to accomplish catalytic C–H functionalization –49. In f2 to polar multiple bonds . In. In2 to le bonds .2 [2 has not been observed [These Roper-type carbenes also reacted with organic azides and nitrous oxide via an apparent [3 + 2] cycloaddition –77, lead2 , and thiδ−═Eδ+ variety can be very useful for inducing atom or group transfer from heterocumulenes or other polar multiple bonds such as those in phosphine oxides. Compared with early-metal Mδ−═Eδ+ FLPs, these sorts of complexes have the advantage of greater redox activity, facilitating application in catalysis (see below). However, they also have lower reactivity due to a less polarized M═E bond, which cannot activate strong C–H or related bonds. Though H–H addition across Ir═C bonds has been reported, this almost certainly occurs by oxidative addition of H2 at the Ir(I) center followed by hydride migration .,87.tert-nylation –89 The uexamined .t-Bu with organic azides leads to the formation of tert-butyl formimidates and (PNP)Ir–N2, which is a suitable precursor for C–H activation of MTBE upon photolysis [AgOTf] is supplied in CIF format and has been deposited with the CCDC, No. 889634.The single-crystal X-ray structure of [(PNP)Ir═C(H)O


Accurate blood pressure monitoring is critical for the management of chronic kidney disease, but changes in management in secondary care clinics may be based on a single blood pressure reading, with a subsequent lack of accuracy. The aim of this study was to evaluate a fully automated sphygmomanometer for optimising the accuracy of blood pressure measurements in the setting of secondary care renal clinics.Patients had routine blood pressure measurements with a calibrated DINAMAP PRO400 monitor in a clinical assessment room. Patients then underwent repeat assessment with a DINAMAP PRO400 monitor and BpTRU device and subsequent 24 hour ambulatory blood pressure monitoring (ABPM).The BpTRU systolic (± SD) reading (117.3 ± 14.1 mmHg) was significantly lower than the routine clinic mean systolic blood pressure  and the repeat blood pressure taken with a DINAMAP PRO400 monitor in a quiet room . The routine clinic mean diastolic (82.4 ± 11.2 mmHg) was significantly higher than the BpTRU reading . Clinic BpTRU measurements were not significantly different to the daytime mean or overall mean of 24 hour ABPM.In patients with CKD, routine clinic blood pressure measurements were significantly higher than measurements using a BpTRU machine in a quiet room, but there was no significant difference in this setting between BpTRU readings and 24 hour ABPM. Adjusting clinic protocols to utilise the most accurate blood pressure technique available is a simple manoeuvre that could deliver major improvements in clinical practice. Chronic kidney disease (CKD) affects up to 15% of the adult population  and is aPatients at highest risk from CKD are managed in secondary care renal clinics; however, the management changes that clinicians recommend in this setting are often based on a single clinic BP reading. Use of a single clinic measurement may over-diagnose hypertension in up to 30% of the general population -13; in mIn addition to the shortfalls of a single BP measurement, inaccuracies may be caused by operator errors; these include incorrect positioning of the patient, inappropriate cuff usage, an inadequate rest period and overly rapid deflation rate ,21-25. IAmbulatory BP monitoring allows BP to be measured over a prolonged period  and is currently considered the 'gold standard’ for the diagnosis of hypertension ,30. An aThe BpTRU device is a fully automated sphygmomanometer that takes six sequential BP readings at pre-set intervals; it automatically discards the first reading and produces an average BP from the last five readings. The BpTRU device has been clinically validated to British Hypertension Society (BHS) A/A grade accuracy . StudiesThe aim of this study was to investigate the clinical utility of the BpTRU device in people with CKD who are attending secondary care clinics. To address this, we have assessed the relationship between i) routinely taken single BP measurements and clinic BpTRU measurements and ii) clinic BpTRU measurements and 24 hour ambulatory BP measurements.This study was registered with the Trust Research & Development at University Hospital Birmingham as part of a clinical service evaluation (CA2-03382-10), and fully complies with the Declaration of Helsinki. The patients were adults attending nephrology outpatient clinics at the Queen Elizabeth Hospital Birmingham. Each patient had a routine clinic BP taken by a clinic nurse in a multi-use clinical assessment room, using a calibrated DINAMAP PRO400 monitor . The DINAMAP PRO monitor series have been validated  and showAfter the routine clinic BP check, patients were approached (by SB) and asked to participate in an assessment of the accuracy of BP measurements. All of the patients who were approached gave informed consent; they were then allocated into one of two groups: the first 45 consecutive patients are reported as 'Group A', a further 25 consecutive patients are reported as 'Group B’. Twenty one consecutive patients attending the outpatient department for ambulatory BP monitoring and who underwent BpTRU evaluation immediately before ambulatory BP monitoring are reported as 'Group C’. A flow diagram summarising the BP measurement schedule is shown in Figure Group A (n = 45) underwent repeat assessment with a separate calibrated DINAMAP PRO400 monitor and then assessment by a BpTRU device  in a quiet clinic room with only the operator present. Of this group, 24 patients had BP re-measured with the DINAMAP PRO400 monitor. They then had BP taken with a one-off reading with the BpTRU monitor. This was followed by a five-minute rest period (consistent with BHS guidelines), and then BP measurement with the BpTRU device, using the full six minute protocol. For BpTRU measurements, the patient was seated (back supported) and the appropriate cuff size, according to measurements on the BpTRU cuffs, was applied to the non-dominant arm (same arm as used for routine clinic BP measurement), at the level of the heart. The BpTRU device is a fully automated sphygmomanometer, which uses the oscillometric technique, and has been clinically validated to BHS A/A accuracy . It is dGroup B (n = 25) underwent immediate repeat assessment in a quiet clinic room with the BpTRU monitor, using the full six minute protocol and with no five minute rest. Group C (n = 21) also underwent immediate repeat assessment with the BpTRU monitor in a quiet room ; the patients in Group C then returned to the clinical assessment room to have their ambulatory BP device  fitted. After fitting the ABPM monitor, an initial BP reading was taken (patients sitting with their back supported and the cuff at the level of the heart). The ABPM device was then set to obtain automatic BP readings in accordance with published guidelines . The ABPDescriptive statistics were calculated for all patients involved in the study (n = 91) and the three groups. Continuous variables are reported as mean ± standard deviation throughout where normally distributed. The analysis focused on comparisons of different BP readings: i) BP measured with a DINAMAP PRO400 monitor in a clinical assessment room as part of routine clinic proceedings; ii) BP measured with DINAMAP PRO400 monitor in a quiet clinic room; iii) BP measured with a BpTRU device in a quiet room, with or without a five minute rest beforehand and iv) BP measured with the ABPM device.post hoc tests were then used to show specific differences between groups. Non-Gaussian data (descriptive statistics only) were analysed with non-parametric Kruskal-Wallis ANOVA tests. Paired Student’s t-tests were used to reveal significant differences between routine clinic and BpTRU BP readings (comparing two different readings only); noted in the relevant Figure legends. Correlation between eGFR and nocturnal ambulatory BP blunting was assessed using Pearson’s correlation coefficient (the data were confirmed as Gaussian prior to using this method). A critical P value of P < 0.05 was considered significant for the statistical tests used throughout this study. Graphically, P < 0.05 is denoted as *, P < 0.01 as **, and P < 0.001 as ***.GraphPad Prism  was used for all statistical analysis. Data were checked for Gaussian distribution prior to further analysis. For data that were normally distributed, we used analysis of variance  to reveal significant differences overall; Bonferroni’s multiple comparisons Ninety one patients were included in the study; 34 female (37.4%) and 57 male (62.6%). The mean age was 49 years (± 18.4), with a range of 16 – 83 years. There was no significant difference in age or gender between the groups.2. The overall mean albumin:creatinine ratio (ACR) was 54.6 ± 146.3 (0 – 1203.6) mg/mmol. There was no significant difference in eGFR or ACR between each group  was 45.1 ± 24.2 (range 4 – 92) ml/ min/1.73 mThe BpTRU systolic reading (122.0 ± 13.9 (96 – 150) mmHg) was significantly lower than the routine clinic systolic BP (149.7 ± 18.5 (117 – 209) mmHg; n = 45; P < 0.0001). The BpTRU diastolic reading (78.4 ± 10.0 (53 - 97) mmHg) was significantly lower than the routine clinic diastolic BP (82.4 ± 11.2 (49 – 100) mmHg; n = 45; P = 0.0033; Figure The mean BpTRU systolic reading (117.3 ± 14.1 mmHg) was significantly lower than both routine clinic systolic (143.8 ± 15.5 mmHg) and systolic BP taken with a DINAMAP PRO400 monitor in a quiet room . The mean BpTRU diastolic reading (74.1 ± 9.2 mmHg) was also significantly lower than both the routine clinic mean diastolic (78.9 ± 12.2 mmHg) and diastolic recorded with a DINAMAP PRO400 monitor in a quiet room  was significantly higher than the mean BpTRU average final reading . However, there was no significant difference between BpTRU systolic readings from Group A  and Group B  was significantly higher than the ABPM daytime mean systolic  and the ABPM overall mean systolic . The routine clinic diastolic reading (90.5 ± 16.2 mmHg) was significantly higher than the ABPM overall mean diastolic value  was not significantly different to either the ABPM daytime mean systolic , or the ABPM overall mean systolic . There was no significant difference between the BpTRU diastolic reading (89.1 ± 16.9 mmHg) and the ABPM daytime mean  or ABPM overall mean diastolic measurements ; 6.28 ± 2.83% for patients with an eGFR from 30-59 ml/ min/ 1.73 m2 (n = 10) and 12.80 ± 8.12% for patients with an eGFR ≥60 ml/min/ 1.73 m2 (n = 6). There was a positive correlation (non-significant) between eGFR and difference between day-time and night-time systolic BP ; the lack of statistical significance may reflect the relatively small numbers who underwent ABPM in the study.Patients in Group C n = 21) underwent routine clinic BP measurements, followed by BpTRU readings, prior to having 24 hour ABPM. The night-time mean ambulatory systolic BP (135.6 ± 20.1 mmHg) was significantly lower than both the day-time  and overall  ambulatory systolic BP readings (denoted as *** in Table 1 underweThis study showed that the use of BpTRU in an outpatient CKD clinic leads to significantly more accurate BP measurement (using 24 hour ABPM as a gold standard) than BP obtained with a conventional BP monitor (DINAMAP PRO400); in this study, there was no significant statistical difference between BP readings taken with BpTRU and daytime and overall 24 hour ABPM. The results show a hierarchy of increasing accuracy from single clinic BP measurements to BpTRU on full protocol; these differences in BP, when measured under different conditions, have important clinical implications and may lead to inaccurate BP management.In addition, we have shown that BpTRU measurements taken in a quiet clinic room are significantly lower than the readings obtained with a DINAMAP PRO400 device in the same setting. We have also demonstrated that, in the presence of an operator, the first BpTRU reading is still significantly higher than the average BpTRU reading.et al. (2011) compared BpTRU readings with the most recent manual routine office BP. Their study also showed that average BpTRU readings were significantly lower than the routine office BP readings [et al. (2008) compared BpTRU readings with mean office manual BP readings taken on the same day. For the office readings in their study, four consecutive readings were taken at one minute intervals; the first reading was then discarded and the mean of the last three readings was recorded. Their results showed that the average BpTRU reading was significantly lower than the mean office BP [A number of primary care studies have investigated the difference between routine office BP measurements and average BpTRU readings. Three studies restricted their subjects to patients with an existing diagnosis of hypertension ,33,34; office BP . Anotherffice BP . The resHowever, there are several differences between this study and previous studies carried out in primary care. Firstly, our measurements were taken in a secondary care outpatient clinic. Secondly, patients in this study were attending for routine appointments, rather than to participate in the study. Thirdly, all the patients had CKD; accurate BP assessment and management is particularly important in this patient group, as people with CKD are at very high risk of cardiovascular disease and hypertension is the major treatable risk factor for this complication. Finally, routine office BP measurements in previous studies have been taken with manual sphygmomanometers; this study compared average BpTRU readings to BP measurements taken with a DINAMAP PRO400 device, an automated sphygmomanometer which, like the BpTRU device, also uses the oscillometric technique.A recent study compared BpTRU readings with routine clinic BP measurements in secondary care clinics . The patSeveral previous studies showing a significant difference between BpTRU and routine clinic measurements have included a five-minute rest prior to carrying out measurements with BpTRU, as per the BHS guidelines ,29,54,60The first BpTRU reading was significantly higher than the average BpTRU reading. This is in agreement with results reported by previous studies ,55,61. INight-time ambulatory BP readings were significantly lower than the daytime and overall ambulatory BP readings. However, a blunted sleep-time decline  in BP was common; a finding consistent with other studies that have assessed people with CKD -35. BlunIn this study, there was no detectable difference between average BpTRU readings obtained in outpatient clinics and the daytime mean or overall mean readings from 24 hour ABPM; again, this is consistent with other published studies ,53-55. BOne shortfall of the study is the absence of 48-hour ABPM data. A recent consensus recommendation concluded that 48-hour ABPM should be utilised for assessment of hypertension, including patients with CKD . The datOther limitations to this study include: i) this study was performed in a clinical setting, rather than a research setting, unlike the majority of previously published work in this area. Therefore, BpTRU readings were always carried out following the routine clinic BP measurements; ii) previous studies have taken routine clinic BP measurements and BpTRU measurements in random order; this is clearly a more accurate way to compare the two techniques, and iii) routine clinic BP measurements were not consistently carried out following a five-minute rest period, unlike those reported in the literature. In a busy outpatient setting, some patients had their routine clinic BP measured as soon as they arrived at clinic; others had their routine clinic BP measured after sitting in the waiting room for a variable amount of time. This lack of control over the technique used for the routine clinic BP measurements may be responsible for some of the difference observed between these readings and the subsequent BpTRU readings. It is also important to emphasise that data from a single study carried out in one setting may not be applicable to other settings. However, in previous studies, where routine clinic BP measurements were carried out in a more controlled manner, average BpTRU readings were also significantly lower than routine clinic readings, suggesting that the data we present are broadly consistent with previous findings and also confirming that previous work carried out in a research setting may be applicable to routine clinical practice.In conclusion, our study demonstrates that routine clinic BP measurements are significantly higher than measurements using a BpTRU machine in a quiet room, and there is no significant detectable difference between BpTRU readings and the day-time mean of 24 hour ABPM in patients with CKD. Adjusting clinic protocols to utilise the most accurate BP device available is a simple manoeuvre that could deliver major improvements in clinical practice.The authors declare that they have no competing interests.SB contributed to the design and co-ordination of the study; undertook data collection and statistical analysis and drafted the manuscript. MD helped co-ordinate the study and reviewed the manuscript. CF critically reviewed the draft manuscript. PC and SS contributed to the conception and design of the study and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2369/14/218/prepub


Oncorhynchus kisutch). Full-size females were included for a sex-specific comparison. A size-specific response in haematology to an acute thermal challenge (from 7 to 20°C at 3°C h−1) was apparent only for plasma potassium, whereby full-size males exhibited a significant increase in comparison with smaller males (‘jacks’). Full-size females exhibited an elevated blood stress response in comparison with full-size males. Metabolic recovery following exhaustive exercise at 7°C was size-specific, with jacks regaining resting levels of metabolism at 9.3±0.5 h post-exercise in comparison with 12.3±0.4 h for full-size fish of both sexes. Excess post-exercise oxygen consumption scaled with body mass in male fish with an exponent of b = 1.20±0.08. Jacks appeared to regain osmoregulatory homeostasis faster than full-size males, and they had higher ventilation rates at 1 h post-exercise. Peak metabolic rate during post-exercise recovery scaled with body mass with an exponent of b∼1, suggesting that the slower metabolic recovery in large fish was not due to limitations in diffusive or convective oxygen transport, but that large fish simply accumulated a greater ‘oxygen debt’ that took longer to pay back at the size-independent peak metabolic rate of ∼6 mg min−1 kg−1. Post-exercise recovery of plasma testosterone was faster in jacks compared with full-size males, suggesting less impairment of the maturation trajectory of smaller fish. Supporting previous studies, these findings suggest that environmental change and non-lethal fisheries interactions have the potential to select for small individuals within fish populations over time.Evidence is building to suggest that both chronic and acute warm temperature exposure, as well as other anthropogenic perturbations, may select for small adult fish within a species. To shed light on this phenomenon, we investigated physiological and anatomical attributes associated with size-specific responses to an acute thermal challenge and a fisheries capture simulation (exercise+air exposure) in maturing male coho salmon ( Evidence exists to suggest that both chronic and acute warm temperature exposure may benefit small animal species and small adult individuals within a species Oncorhynchus tshawytscha) suggested that small individuals may be more tolerant of an acute thermal challenge than larger conspecifics For fishes, climate change and variability are altering species distributions as populations respond to warming waters e.g., , and theAn additional challenge faced by many fish species is the increasing likelihood of interaction with commercial or recreational fisheries. Fisheries capture may result in direct mortality, although fish commonly escape following contact with fishing gear, or they are intentionally released as bycatch or through catch-and-release fisheries. Fishes can encounter similar stressors under natural conditions, such as during non-lethal interactions with animal predators . Released or escaped fish must physiologically recover from the encounter or risk mortality Oncorhynchus kisutch) was chosen as the model species for several reasons: (1) freshwater and marine ecosystems utilised by coho salmon have warmed, and fish now have a greater probability of acutely encountering higher river temperatures than at any point since records began in the 1940s Given the sparse dataset concerning these issues, the present study sought to explore a range of physiological and anatomical attributes that might benefit small individuals within a species when exposed to an acute thermal challenge or a simulated fisheries/predator encounter. Attention was focussed on the oxygen transport system and blood chemistry, as these have been previously implicated in determining the tolerance of fishes to various environmental challenges Oncorhynchus kisutch) were dip-netted throughout October and November in 2009 and 2010 as they completed their 140 km upstream migration from the Pacific Ocean to their natal rearing location at the Chehalis River Hatchery , where they had been released previously as one year old fish. Following their first year in freshwater, most individuals spend 1.5–2.5 years foraging in the marine environment prior to returning to their natal freshwater rearing location as 2.5–3.5 year-olds , yet some individuals  mature and return to the rearing location after only 6 months in the ocean and at a much smaller body size . Full-size males (2.5–3.5 years old) and precocious males  were used in the present study to investigate the effects of body size . Full-size females were also used to examine for sex-specific differences in full-size fish. Precocious females can occur but are extremely rare, and none were encountered throughout the two years in which the present study was conducted. While it is generally unavoidable to have age and size covarying when examining intraspecific body mass (Mb) scaling in fishes, we believe that the male coho salmon model used here is better than most because the fish are not feeding/growing and they are at similar stages of reproductive maturity. All experiments were conducted with the approval of the Animal Care Committee of the University of British Columbia, in accordance with the Canadian Council on Animal Care.Mature coho salmon  than full-size fish when exposed to an acute thermal challenge. Coho salmon  were placed individually into opaque holding tubes equipped with plastic mesh at each end to allow good flow-through of water. The tubes were either 90×22 cm (length×diameter) for full-size fish or 70×12 cm for jacks. The tubes were evenly (or near-evenly) distributed between two enclosures, both containing flow-through river water at ambient temperature (∼7°C). The tubes were arranged side-by-side and also stacked 2–3 high, such that all fish within each enclosure were housed within a space of ∼1.5 m−1 from 7 to 20°C, after which the fish were maintained at 20°C for a further 2 h. At this point, all fish from the control enclosure (7°C) and warm enclosure (20°C) were individually removed from the holding tubes, sacrificed by cerebral concussion, and sampled for blood (∼3 ml) from the caudal vasculature using lithium-heparinised vacutainers. Extreme care was taken to avoid disturbing each individual until <30 s before it was removed and sampled for blood, as this is known to provide a blood sample that is uninfluenced by the handling event All fish were given 20 h to recover from handling before the group in one enclosure (warm) was exposed to an acute thermal challenge while the group in the second enclosure (control) remained at ambient temperature and acted as a control. A gas-powered water heating system was used to increase the temperature of the warm enclosure by 3°C hMb ranges and sample sizes  of: full-size males, Mb = 1.39–4.22 kg, N = 9 and Mb = 1.39–3.71 kg, N = 7; full-size females, Mb = 2.06–3.35 kg, N = 9 and Mb = 1.31–3.93 kg, N = 8; jacks, Mb = 0.20–0.51 kg, N = 7 and Mb = 0.22–0.60 kg, N = 7.Three identical trials were conducted on separate days, giving total −1; ∼7°C). The chasing protocol rendered all individuals unable to continue burst swimming after about 2 min. Sampling took place at 1, 2, 4, 8 or 19 h post-treatment, when individual fish were rapidly removed from the recovery box, sacrificed by cerebral concussion and sampled for blood by caudal puncture as described above. Full-size fish (both sexes) and jacks were examined. Ten recovery boxes were used in each trial, and trials were repeated to ensure appropriate sample sizes in each size-grouping and sex-grouping, and at each post-treatment recovery period (sample sizes in figures). Ventilation rate was measured in a subsample of fish  at 1 h post-exercise through a small viewing slit in the lid of each recovery box.The exhaustive exercise protocol was identical to that used previously min and peak, respectively) were taken as the minimum and maximum values obtained for each individual throughout the 19 h recovery period, with the former always occurring after the latter.Additional fish  underwent the same treatment protocol as above but were placed individually and immediately into static respirometers (each 138 l) rather than recovery boxes during the post-treatment recovery period. This group of fish was not sampled for blood, but rather remained in the respirometers for 19 h while the rate of oxygen consumption  was surgically implanted with biologgers and given 6 d to recover before undergoing the exhaustive exercise protocol described above. Jacks were not used as they were too small to receive a biologger. Surgical methods were identical to those used previously g for 7 min. Remaining blood was spun at 7,000×g for 7 min and then the plasma was collected in cryogenic vials and stored in liquid nitrogen prior to being transferred to a −80°C freezer for subsequent analyses.Blood samples were immediately placed into an ice slurry and processed within 20 min. Haematocrit (Hct) of whole blood was measured using micro-capillary tubes spun at 10,000×www.ysilifesciences.com). Plasma measurements were made in duplicate of cortisol , osmolality (Advanced Instruments 3320 freezing point osmometer), chloride , sodium and potassium  , an incision was made around the perimeter of the opercular cavity and the entire gill basket was removed, rinsed, blotted and weighed. For six fish , a section of gill was removed immediately after death  and fixed in 10% acetate-buffered formalin. The gill samples were subsequently dehydrated in an ethanol series and embedded in paraffin wax, before being sectioned at 5–8 µm and stained with Masson's trichrome. Images were taken for histological comparisons using an Olympus Provis AX70 microscope  in combination with a AxioCam HRc cooled CCD camera . AxioVision software  was used to measure the following variables. Blood-water diffusion distance, maximum erythrocyte diameter , and gill lamellae diameter were measured in images taken at 40×magnification. Gill lamellae length and interlamellar distance were measured in images taken at 10×magnification, while gill filament diameter was measured in images taken at 4×magnification (full-size fish) or 10×magnification (jacks). Ten measurements of each parameter were taken at random for each fish in areas where the gill filaments were least variable in diameter, distal from the gill arch.www.systat.com) using Bonferroni correction where necessary to account for multiple comparisons. All blood data for between group comparisons were log-transformed prior to statistical analyses to satisfy tests for normality. Statistical differences resulting from the thermal challenge were assessed using two-way ANOVA with group  and temperature as factors, including the interaction term group*temperature. Two-way repeated measures ANOVA was used to investigate differences in Statistical tests were performed in SPSS  and SigmaStat  and potassium (13.3 mmol l−1) were higher in this fish than in any other individual, while plasma testosterone (2.2 ng ml−1) was the lowest of all fish.Increasing water temperature to 20°C had a significant effect on the blood properties of all groups of fish in comparison with the respective control groups held at 7°C , the initial increase in −1; 0.40±0.03 kg) than in full-size fish .The exercise and air exposure protocol caused complete exhaustion in all groups of fish (no capacity to continue burst swimming) and a significant increase in  . The  reThe exercise protocol caused an abrupt increase in heart rate of full-size fish (both sexes), which progressively decreased to resting levels over a longer time period than Blood variables clearly illustrated the stress response linked with the exhaustive exercise protocol, such as increases in Hct, plasma lactate and cortisol, and disruption to plasma ion concentrations . Plasma me point . One of me point .b) were 1.05±0.06 for minimum min; taken as the lowest peak; taken as the highest peak/min) did not differ substantially with increasing body mass. Excess post-exercise oxygen consumption  was disproportionately higher in full-size males than in jacks  rather than any size-specific differences in mass-specific min or peak.Physiological and morphological data were compared across all males, which spanned over an order of magnitude in body mass . The masb = 1.05–1.07; b = 1.13±0.05). Gill histology revealed size-specific differences in gill filament diameter (b = 0.29±0.06), lamellae length (b = 0.34±0.12), and interlamellar distance  to examine for any scaling of blood variables  was related to group differences  rather than body mass per se. Nevertheless, this finding should be taken with caution due to low sample size at 20°C  to provide a large dataset  than did a Ucrit test peak in this study are only slightly lower than the values of maximum While it has been proposed that the phenomenon of size-specific thermal tolerance in adult fishes is linked with a decrease in aerobic scope with increasing body mass e.g., , the ress at 7°C . It shouO. nerka) tended to have lower migration survival than smaller individuals when released in the marine environment en route to spawning grounds following a fisheries capture event b = ⅔) and the latter is a mass . Based on the above points, and the higher ventilation rate of jacks at 1 h post-exercise, it may seem reasonable to assume that metabolic recovery of full-size fish may have been limited by oxygen uptake at the gills rather than by convective oxygen transport through the circulatory system. However, isometric scaling of peak, constant blood-water diffusion distance across body mass, and expected scaling  of gill filament diameter (b = 0.29) and lamellae length (b = 0.34) all suggest that there was no size-specific effect on the rate of either diffusive or convective oxygen transport.Despite the subtle size-specific responses of coho salmon to acute warming, clear size-specific differences in metabolic recovery from exhaustive exercise were apparent . Mass-sppeak of ∼6 mg min−1 kg−1. Regaining osmoregulatory homeostasis is a contributor to the oxygen debt recovery and jacks appeared to accomplish this faster than full-size males following the exhaustive exercise protocol (A critic could argue that jacks did not exhaust themselves as much as full-size fish during the exercise protocol, which subsequently caused the size-specific differences in metabolic recovery. However, this is highly unlikely since jacks appeared equally exhausted as full-size fish during and following the protocol, and plasma lactate and cortisol reached similar levels independent of fish size . Indeed,protocol . PreviouAdding to the known effects of stress on reproductive maturation and gamete quality e.g., , the preFisheries have been implicated in modifying the size structure of fish populations by harvesting larger individuals and avoiding or releasing smaller ones


Interferon-beta (IFNβ) is used to inhibit disease activity in multiple sclerosis (MS), but its mechanisms of action are incompletely understood, individual treatment response varies, and biological markers predicting response to treatment have yet to be identified.The relationship between the molecular response to IFNβ and treatment response was determined in 85 patients using a longitudinal design in which treatment effect was categorized by brain magnetic resonance imaging as good (n = 70) or poor response (n = 15). Molecular response was quantified using a customized cDNA macroarray assay for 166 IFN-regulated genes (IRGs).The molecular response to IFNβ differed significantly between patients in the pattern and number of regulated genes. The molecular response was strikingly stable for individuals for as long as 24 months, however, suggesting an individual ‘IFN response fingerprint’. Unexpectedly, patients with poor response showed an exaggerated molecular response. IRG induction ratios demonstrated an exaggerated molecular response at both the first and 6-month IFNβ injections.MS patients exhibit individually unique but temporally stable biological responses to IFNβ. Poor treatment response is not explained by the duration of biological effects or the specific genes induced. Rather, individuals with poor treatment response have a generally exaggerated biological response to type 1 IFN injections. We hypothesize that the molecular response to type I IFN identifies a pathogenetically distinct subset of MS patients whose disease is driven in part by innate immunity. The findings suggest a strategy for biologically based, rational use of IFNβ for individual MS patients. Development of type I IFN therapy continued, however, leading to IFNβ approval.IFNβ (a type I IFN) treatment is therapeutic in MS patients, as indicated by clinical trial patient group results. However, results from a clinical trial of interferon gamma (IFNγ) (a type II IFN) Increased bioavailability of type I IFNs is involved in diverse autoimmune diseases Poor clinical response to IFNβ can be related to production of IFNβ-neutralizing antibodies or to genetic variants in IFNβ receptors or signalling components The Cleveland Clinic Institutional Review Board approved the study. After discussion with our bioinformatics colleagues, but without a formal power calculation, we sought to enroll 100 patients. All subjects provided written informed consent. Subjects were eligible if they had clinically isolated syndrome (CIS) or relapsing-remitting MS, were treatment-naïve, were initiating intramuscular IFNβ-1a treatment, and were followed at the Mellen Center (our MS center). Each patient was examined at baseline, 6, 12, and 24 months. At 3 and 18 months, patients were contacted by phone to assess treatment compliance and ascertain side effects. At baseline, 6, and 24 months, blood was collected for IRG analysis in a clinical research unit immediately before and exactly 12 hours after an IFNβ injection, and the patients had standardized brain MRI scans for quantitative assessment of lesions and brain atrophy. At each visit, patients had neurological exams to determine the Kurtzke Expanded Disability Scale Score 3, the lesion must grow by 20% to be counted as enlarging. For lesions <150 mm3, the lesion must grow by 50% to be counted as enlarging. New and enlarging T2 lesions were visually verified using interactive software to generate the final counts.The MRI acquisition included T2-weighted fluid-attenuated inversion recovery (FLAIR), T2- and proton density-weighted dual echo fast spin echo, and T1-weighted spin echo images acquired before and after injection of a standard dose of gadolinium (0.1 mmol/kg). Images were analyzed using software developed in-house to determine brain parenchymal fraction (BPF), T2 lesion volume, gadolinium-enhancing lesion volume and number, and the numbers of new and enlarging T2 lesions. BPF was calculated from FLAIR images using fully automated segmentation software ex-vivo from blood using the PAXgene RNA blood extraction kit  per the manufacturer's instructions and concentrated by ethanol precipitation. RNA quality and quantity were assessed by spectrophotometry  and visualized by agarose gel electrophoresis. Samples were stored at −80°C.RNA was extracted The cDNA macroarray analysis was performed as previously described ex-vivo from blood was used for generating radiolabeled cDNA probes by reverse transcription with Superscript II in the presence of 32P-dCTP . Residual RNA was hydrolyzed by alkaline treatment at 70°C for 20 min after which cDNA was purified using G50 columns . Preparation of macroarrays and hybridization of radioactive cDNA were conducted as described previously The protocol for spotting DNA on the membrane, probe labeling, and hybridization has been described previously, with modifications as follows To minimize variability, each patient's samples at baseline and 6 months were processed in a single batch experiment. A detailed laboratory protocol for the macroarray method is available on request.IRs were validated using real-time (rt) quantitative PCR for 5 genes: OASL (accession number NM003733); TRAIL (U37518); IFI44 (D28915); HLADRA (J00194); and TIMP-1 (M59906). Spearman correlation coefficients between IRs calculated from rt-PCR and macroarray data for OASL, TRAIL, IFI44, HLADRA, and TIMP-1 were 0.92, 0.75, 0.36, 0.72, and 0.54, respectively, all statistically significant.Poor response to IFNβ was based on quantitative MRI analysis, comparing the MRI at the 6-month visit with baseline. Poor response was defined as the occurrence of ≥3 new or enlarging lesions. Differences in baseline characteristics between good responder (GR) and PR groups were compared using t-tests or Fisher's exact tests, as appropriate. A Poisson regression was used to test group differences in the number of induced IRGs with IRs≥2.0 at the baseline injection. Pearson correlation coefficients of log2 transformed IRs at initial injection compared with 6 months were computed for all 85 patients. Baseline, 6-month, and 24-month pair-wise correlations were computed for 5 randomly selected patients from each group.To minimize noise, a filter was applied to exclude IRGs that were not regulated in this study. Genes with mean IRs within the range of 0.9 to 1.1  were excluded. Using this method, 48 of the 166 IRGs were eliminated from further analysis at the initial IFNβ injection, and 30 IRGs were eliminated at the 6-month injection. For the remaining IRGs, covariate-adjusted least-square means (LS means) of the log2-transformed IRs were computed and compared between response groups by ANCOVA. The covariates were baseline age, sex, presence of gadolinium-enhancing lesions, and T2 volume.The remaining IRGs were classified as upregulated or downregulated as follows: an IRG was upregulated if more than 50% of the 85 subjects had an IR>1.0; an IRG was classified as downregulated if more than 50% had an IR<1.0. For each IRG, LS means of the IRs were compared between PRs and GRs. The response was classified as exaggerated at the group level if PRs>GRs in upregulated genes, or PRs<GRs in downregulated genes. The proportion of genes showing an exaggerated response in PRs was tested (one-sided) with a binomial proportion test assuming a null hypothesis of proportion≤0.5.To further investigate whether IRGs could discriminate between the responder groups, the magnitude of the exaggeration in each patient was computed as a sum of the exaggerated amounts  for each regulated IRG. That is, for upregulated genes, if the individual IR was greater than the median for the whole group, or for downregulated genes, if the individual IR was less than the median for the whole group, then the exaggerated amount was calculated. The magnitudes of exaggerated responses were compared in PRs and GRs at the first and 6-month injections using ANCOVA with baseline demographic features, presence of baseline gadolinium lesions, and baseline T2 lesion volume as covariates..Of the 99 subjects enrolled, 85 continued to take intramuscular IFNβ-1a for at least 6 months, which was the predetermined time-point for determining treatment response based on MRI, for correlation with IRG macroarray results. Reasons for drop out included: side effects (n = 4); new health issues (n = 2); no explanation provided (n = 4); left area (n = 1); switched to glatiramer acetate per patient preference (n = 1); sample hybridization not adequate at baseline or 6 month injection (n = 2). Subjects were encouraged to remain in the protocol for 24 months, so that the relationship between 6-month MRI activity and later disease progression, as well as stability of the IFNβ molecular response, could also be determined. At the time of this report, subject accrual was complete, and all subjects had 6-month visits. This report focuses on the analysis of 85 patients with complete data at baseline and 6 months. Of the 14 patients who were enrolled in the study but did not complete the planned 6-month macroarray analysis, 12 discontinued IFNβ-1a, whereas sample hybridization was unsuccessful in the other 2, either at first injection or 6 months. Ten patients who had completed the 24-month protocol contributed blood for 24-month IRG analysis. Baseline demographic and disease characteristics did not significantly differ between those who completed the first 6 months and those who did not (data not shown). For all other analyses, only the 85 patients who completed the first 6 months were included. Among these 85, 32% had CIS with multiple brain MRI lesions, and 68% had relapsing-remitting MS. The mean age was 35.7 years; mean disease duration was 2.4 years; 65% were women; and 91% were white. At 6 months, 15 (18%) subjects were classified as PRs based on our pre-determined MRI definition. The two groups were similar at baseline on all characteristics except that a higher proportion of PRs had gadolinium-enhancing lesions and greater T2 lesion volumes at baseline, indicating PRs had more severe disease .22.Preliminary assays in healthy subjects not receiving IFNβ showed that IRs did not vary more than 1.5-fold in assays separated by 12 or 24 hours. Therefore, an IR≥2.0 defined induction of an IRG. The number of induced IRGs at the first IFNβ injection varied among patients, ranging from 7 to 135, with no relationship between IFNβ responder status and number of induced genes (p = 0.76) (data not shown). Similarly, the pattern of response to the initial IFNβ injection varied considerably between patients as previously reportedExcluding those three subjects, IRs at first injection strongly correlated with IRs at 6 months for individual patients [Pearson correlation coefficient mean (±SD) = 0.81±0.11]. The mean correlation coefficient for the 15 PRs  was 0.81±0.10, compared with a mean of 0.81±0.11 for the 67 GR patients ; between 6 months and 24 months ; and between baseline and 24 months . The IRG analysis was repeated at 24 months for 10 selected patients (5 PRs and 5 GRs) . The IRThe biological effects of IFNβ are traditionally accounted for by the activities of the IRG protein products Although IFNβ is the most commonly used disease-modifying treatment for MS, its mechanisms of action are not fully understood, no biological markers are available to guide individualized therapy, and differential clinical responsiveness is not understood at a mechanistic level. We sought to determine molecular correlates of the treatment response to IFNβ therapy in relapsing-remitting MS. Response to IFNβ was defined in advance, using MRI to classify treatment response in individual patients. In that regard, numerous reports have documented that patients developing new lesions while on IFNβ have relatively poor clinical outcomes. We initially hypothesized that 1) the molecular response to IFNβ would differ between patients but would be stable over time within individuals, and 2) treatment response would correlate with the nature of the primary molecular response or its persistence over time. We confirmed that the molecular response differs significantly between patients in regard to the identity and number of regulated IRGs and the extent of induction or repression.This study differs methodologically from prior studies of IRG expression in MS. First, we used a prospective design and a defined, validated outcome measure based on an objective MRI criterion The consistent pattern of exaggerated induction or repression of IRGs in patients who responded poorly was observed at the initial IFNβ injection and confirmed at 6 months, indicating that this exaggerated response was a stable phenotype. Over time, circulating leukocyte subsets change with IFNβ therapy, so it was not surprising that the specific genes identified as distinguishing good and poor responders differed at the two time points. This observation leads to a novel hypothesis: patients with MS who exhibit an exaggerated response to exogenous IFNβ have both a poor treatment response and more severe disease. Indeed, patients categorized as poor responders were more likely to have gadolinium enhancing lesions at baseline, and had a higher T2 lesion burden. Thus, patients with poor response to IFNβ injections already had more active disease at treatment start. We believe the explanation for this observation is that the response to exogenous IFNβ injections reflects responses to endogenous type 1 IFN. It has been well recognized that some, but not all, MS patients experience relapses during viral infection, which strongly induces endogenous type 1 IFN. An exaggerated response to type 1 IFN plus disease driven by aberrant innate immunity might explain the response to viral infection in a subset of MS patients. Our hypothesis would predict that patients experiencing disease activation with viral infections would have more severe disease. This has not been studied.Our study has several limitations. First, there was no placebo-treated, or untreated comparison group, so the subset we’ve identified could simply represent more severe disease, and the outcome might have nothing to do with IFNβ treatment. Second, advances in microarray technology during the course of this longitudinal study now permit more quantitative assessment of the RNA we collected. Those experiments, currently ongoing, have the potential to confirm or refute the current findings, and possibly to extend them considerably. Third, the classification of poor treatment response was based entirely on a prospectively defined MRI outcome. The clinical outcome for poor and good response subgroups needs to be established in long-term follow-up studies, which are ongoing. Lastly, our findings must be confirmed in separate patient cohorts and by independent investigators.H1 cells, but exacerbated disease caused by transfer of TH17 cells. Intriguingly, two IRGs we found to be markedly regulated by IFNβ  would be predicted to act in a manner consistent with the hypothesis proposed by Axtell et al. Specifically, we observed exaggerated inhibition of IL2 and exaggerated induction of PI3K, which together could cause TH17-mediated inflammation that would be resistant to inhibition by gene products of the type I IFN response, including IL10 Our hypothesis is consistent with a recent report by Axtell and colleagues suggesting that fundamentally different pathogenic pathways in MS subjects underlie the differential response to IFNβ therapy Despite these recent reports, predictive biomarkers for IFNβ treatment failure have not been identified or validated that is characterized in part by their response to endogenous type 1 interferon. A corollary is that differences in innate immunity, either within type I IFN pathways or affecting the expression levels of IRGs indirectly, are determinant for enhanced disease severity in PRs.A more wide-ranging implication relates to MS pathogenesis. Mechanistic proposals for disease pathogenesis have focused on adaptive immunity, particularly immune responses directed against myelin constituents. We found that IFNβ recipients who became PRs already had higher levels of disease activity and disease burden, as measured by MRI, upon entering the study. We hypothesize that these patients exhibit a pathogenic pathway 32 or through augmented responses from an equivalent pre-injection baseline. In either case, the findings in this study implicate innate immunity in MS pathogenesis from its onset and may provide novel insights into the fundamental disease process, along with new therapeutic targets and hope for personalized use of IFNβ.In that regard, IFNβ injection can be viewed as a provocative test that may expose a genetically or epigenetically determined type 1 IFN response that contributes to pathogenesis. In these individuals, exogenous IFNβ elicits an exaggerated response, either by upregulation from a lower pre-injection IRG expression levelFigure S1AScatter plots showing the IFNβ molecular response at baseline and 6 months for 57 of 85 patients. The study subject number is indicated above each plot; response at baseline is shown on the x-axis and at 6 months is shown on the y-axis. For each subject, the induction ratio for each of 166 genes is shown at the two time points. The variability of the molecular response between the two time points is indicated by deviation from the diagonal line in each plot. A consistent response at the two time points is evident except for patients 7, 21, and 25 (see text). Also, response consistency was similar between patients with poor treatment response (circled study subject numbers).(EPS)Click here for additional data file.Figure S1BScatter plots showing the IFNβmolecular response at baseline and 6 months for the remaining 37 of the 85 patients. The study subject number is indicated above each plot; response at baseline is shown on the x-axis and at 6 months is shown on the y-axis. For each subject, the induction ratio for each of 166 genes is shown at the two time points. The variability of the molecular response between the two time points is indicated by deviation from the diagonal line in each plot. Response consistency was similar between patients with poor treatment response (circled study subject numbers).(EPS)Click here for additional data file.Figure S2Scatter plots for 10 individual patients showing a consistent response over 24 months. Five good and 5 poor responders were randomly selected from each group with macroarray data at baseline, 6 months, and 24 months. The first 3 columns show patients with a good treatment response, and the last 3 columns are patients with a poor treatment response. Columns 1 and 4 compare responses at baseline and 6 months; columns 2 and 5 compare responses at 6 and 24 months; and columns 3 and 6 compare responses at baseline at 24 months. Note the consistency of response for all time points and that the molecular response is consistent regardless of whether treatment response is good or poor.(EPS)Click here for additional data file.Table S1(DOC)Click here for additional data file.


Bovine serum albumin (BSA) contains high affinity binding sites for several endogenous and exogenous compounds and has been used to replace human serum albumin (HSA), as these two compounds share a similar structure. Naringin palmitate is a modified product of naringin that is produced by an acylation reaction with palmitic acid, which is considered to be an effective substance for enhancing naringin lipophilicity. In this study, the interaction of naringin palmitate with BSA was characterised by spectroscopic and molecular docking techniques.SVK) was found to decrease with increasing temperature, suggesting that a static quenching mechanism was involved. The changes in enthalpy (ΔH) and entropy (ΔS) for the interaction were detected at −4.11±0.18 kJ·mol−1 and −76.59±0.32 J·mol−1·K−1, respectively, which indicated that the naringin palmitate-BSA interaction occurred mainly through van der Waals forces and hydrogen bond formation. The negative free energy change (ΔG) values of naringin palmitate at different temperatures suggested a spontaneous interaction. Circular dichroism studies revealed that the α-helical content of BSA decreased after interacting with naringin palmitate. Displacement studies suggested that naringin palmitate was partially bound to site I (subdomain IIA) of the BSA, which was also substantiated by the molecular docking studies.The goal of this study was to investigate the interactions between naringin palmitate and BSA under physiological conditions, and differences in naringin and naringin palmitate affinities for BSA were further compared and analysed. The formation of naringin palmitate-BSA was revealed by fluorescence quenching, and the Stern-Volmer quenching constant (In conclusion, naringin palmitate was transported by BSA and was easily removed afterwards. As a consequence, an extension of naringin applications for use in food, cosmetic and medicinal preparations may be clinically and practically significant, especially in the design of new naringin palmitate-inspired drugs. Naringin  is a flavonone glycoside  that is Serum albumins are the major components of plasma proteins in all mammals BSA is composed of a single-chain 583 amino acid globular nonglycoprotrein that is cross-linked with 17 cystine residues (8 disulfide bonds and 1 free thiol) K), the number of binding sites (n), thermodynamic parameters for the reaction, and changes to the binding sites and conformation in BSA in response to naringin palmitate were performed. The fact that these are the first spectroscopic results for naringin palmitate-BSA interactions illustrates the complex nature of this subject. A comparison between naringin palmitate-BSA and naringin-BSA was performed in order to confirm naringin palmitate functionality.The induction of the palmitic acid chain by naringin would change the structure and solubility of naringin. The biological and pharmacological functions of naringin palmitate may be different from naringin. In this study, naringin palmitate was been synthesized in organic solvent. It was studied using fluorescence spectroscopy, UV absorption spectroscopy, FT-TR, CD spectroscopy, site maker displacement and molecular modeling methods. Investigations of the binding constant (Candida antarctica) was purchased from Sigma (Germany). Naringin was sourced from Fluka . Palmitic acid  was used as an acyl donor. The tert-amyl alcohol (Merck) was used as solvents during esterification. Molecular sieves of 4 Å (8–12 mesh beads), warfarin and ibuprofen were purchased from Sigma (United States of America). Bovine serum albumin was purchased from Sino-American Biotechnology Company  and used without further purification. The stock solution of naringin and naringin palmitate (5×10−3 M) was prepared in dimethyl sulfoxide (DMSO). Stock solutions of warfarin(1.5×10−3 M) and ibuprofen (1.0×10−3 M) were prepared −5 M) was prepared in 0.1 M NaCl solution. The buffer solution (pH 7.40) was composed of Tris-HCl (0.1 M). All stock solutions were kept in the dark at 4°C and then diluted to the required experimental sample concentrations. All BSA samples were prepared in the pH 7.40 buffer solution. The concentrations of BSA, warfarin and ibuprofen in the spectroscopic samples were determined by UV absorbance measurements using an extinction coefficient of 43800 M−1 cm−1 at 280 nm for BSA −1cm−1 at 308 nm for warfarin −1 cm−1 at 308 nm for ibuprofen Immobilized lipase Novozyme 435  and palmitic acid (250 mM) were mixed in 5 mL of solvents with addition of 4 Å molecular sieves (100 g/L) which were activated by heating at 180°C for 1.5 h prior to use. Naringin acylation was started with the addition of immobilized lipase. Parallel experiments were carried out at a predetermined time interval, and one vial was withdrawn to determine the concentrations of substrate and products by high performance liquid chromatography (HPLC). The reaction was terminated by adding 5 mL of methanol and removing the immobilized enzyme and molecular sieves by filtration. After the reaction was terminated, the naringin palmitic acid esters were purified by column chromatography   on silica gel 60 A, using a mixture of ethylacetate/methanol/methanoic acid  as eluents.All fluorometric experiments were recorded on an F-2500 spectrofluorometer  using a 1 cm quartz cell and a thermostat bath. Ten millilitres of 1.5 µM BSA solution was prepared and titrated with increasing concentrations of naringin and naringin palmitate (0 to 50 µM) in 5 mM buffer solution at three different temperatures . An appropriate blank that corresponded to the buffer was subtracted in order to correct for background fluorescence. The excitation wavelength was 282 nm, and the emission spectra were recorded from 300 to 450 nm. The widths of both the excitation and the emission slit were set to 5.0 nm. The maximum emission intensities were used to calculate binding constants, occupation of binding sites and thermodynamic parameters.0F and F are the relative fluorescence intensities in absence and presence of the quencher, 0τ is the lifetime of the fluorophore in absence of quencher with a value of 10−8 s qk is the quenching constant of bimolecular. SVK is the Stern-Volmer quenching constant and [Q] is the quencher concentration. SVK was obtained by plotting 0/FF versus [Q], and if SVK is much larger than 2×1010 M−1 s−1, then the quenching is static in nature Fluorescence quenching is the decrease of quantum yield of fluorescence from a fluorophore that is induced by a variety of molecular interactions with a quencher molecule K is the binding constant for a site and n is the number of bindings per albumin, respectively. The plot of 0–F)/F0)lg , ΔH and ΔS are the changes in enthalpy and entropy during quenching process.Interaction forces between quencher and biomolecules may include hydrophobic force, electrostatic interactions, van der Waals interactions, hydrogen bonds, and others G) associated with the interaction of naringin/naringin palmitatewith BSA can be calculated from the following equation:The free energy change  were recorded with increasing concentrations of naringin/naringin palmitate (0 to 50 μΜ), by setting Δλ = 60 nm and Δλ = 15 nm for tryptophan and tyrosine residues respectively Synchronous fluorescence spectra of BSA  or ibuprofen (1.0 µM) and BSA and then allowed to stand for 1 hour at 288 K The absorption spectra were measured on a U-3010 spectrophotometer  with a 1.0 cm path length cell. The UV/vis absorption spectra were obtained by scanning the solution containing 1.5 µM BSA solution while increasing the concentrations of naringin and naringin palmitate (0 to 50 µM) in the spectrophotometer with the wavelength range of 295–495 nm. Analyses were carried out at room temperature.MRE) in deg cm2 dmol−1, which can be estimated with Eq. 8 oθ is the observed CD value, pC is the molar concentration of the protein (1.5 µM), n is the number of amino acid residues in the protein (583) and l is the path length of the cell (2 mm). The α-helical contents of free and complexed BSA were calculated from the mean residue ellipticity values at 208 nm using the following equation and as described previously All BSA far-UV CD spectra with different amounts of naringin/naringin palmitate were obtained with a MOS-450 spectropolarimeter  in cylindrical cells (2 mm path length) at 288 K. The concentration of BSA in the CD study was 1.5 µM in 5 mM Tris-HCl buffer with a pH of 7.4. Seven sets of solutions were prepared with BSA and naringin/naringin palmitate in the following proportions 1∶0, 1∶2, 1∶4, 1∶6, 1∶8, 1∶10 and 1∶15. The spectra were recorded from 190 to 250 nm with step resolutions of 0.5 nm, an acquisition duration of 1 s, bandwidth of 5.0 nm and sensitivity of 100 mdeg during all measurements. The spectra in the far-UV (190–250 nm) region required an average of six scans. The spectrum of the buffer and naringin/naringin palmitate control solution was subtracted from the sample spectra. The CD measurements were expressed in terms of mean residue ellipticity  was carried out on a Tensor 37 spectrometer  that was equipped with a DTGS detector, a KBr beam splitter, and a Ge attenuated total reflection (ATR) accessory at room temperature. A 128-scan interferogram with a 4 cmhttp://davapc1.bioch.dundee.ac.uk/prodrg/index.html). Molecular docking simulations of naringin and naringin palmitate were performed with the AutoDock 4.2 program. Each grid computation was performed with a grid box of 60×60×60 Å with 0.375 Å spacing, which covered all the active site residues and allowed for the flexible rotation of the ligand. For rigid docking simulations, the parameters were set to 100 GA runs that terminated after a maximum of 2,500,000 energy evaluations, and the population size was set to 150 with a crossover rate of 0.8 (Lamarkian Genetic Algorithm). For flexible docking, the above parameters were the same, while some important neighbouring residues at the binding sites were set to flexible. The lowest energy conformation in the largest cluster of each docking simulation was extracted and analysed. The hydrogen bonds and hydrophobic interactions between the ligands and the protein were represented with Ligplot version 4.5.3 The crystal structure of BSA was obtained from the Protein Data Bank (entry code 4F5S) The increasing concentration effects of naringin and naringin palmitate (0 to 50 μΜ) on BSA fluorescence intensity at 288 K is shown in F0/F and the concentration cn was obtained. The Stern-Volmer equation values for naringin and naringin palmitate at different temperatures  are shown in To illustrate the differences in BSA fluorescence quenching mechanism between naringin and naringin palmitate, the fluorescence spectra data were analyzed using the Stern-Volmer equation (Eq. 1) SVK) for naringin and naringin palmitate, which were inversely correlated with temperatures. Generally, dynamic quenching and static quenching exist within fluorescence quenching according to the difference of fluorescence quenching way SVK increased with increasing temperature, while the static quenching constant SVK decreased with increasing temperature qK of different quencher with biopolymer was 2×1010 L·mol−1·s−14 L·mol−1 (288 K) 4 L·mol−1 and 1.34×105 L·mol−1 (298 K) On the other hand, the maximum collision quenching constant SVK of naringin palmitate was lower than in naringin /Flg. The binding constants K and binding sites n for naringin and naringin palmitate with BSA were calculated using Eq. 2, and the results are listed in n that were approximately equal to 1 corresponded to the presence of a single binding site in BSA H) and entropy (ΔS) values were obtained from linear Van’t Hoff plots . As H and ΔS for the binding reaction between naringin palmitate and BSA were found to be −4.11±0.18 kJ·mol−1 and −76.59±0.22 J·mol−1·K−1, and the ΔH and ΔS for the binding reaction between naringin and BSA were found to be −13.64±0.46 kJ·mol−1 and 47.23±0.67 J·mol−1·K−1. The negative ΔG corresponded to a spontaneous binding process According to the binding constants that were found at three temperatures above , the change in enthalpy , rather than the change in enthalpy (ΔH), provided a major contribution to ΔGS. This interaction was more likely hydrophobic, with electrostatic interactions involved in the naringin-BSA binding process The interaction forces between quencher and biomolecules may include hydrophobic force, electrostatic interactions, van der Waals interactions, hydrogen bonds, and others H and ΔS  [24]. Thnd ΔS , the critical distance when the efficiency of energy transfer is 50%, the efficiency of energy transfer (E), and the distances between the donor and acceptor (r) were calculated with Eq. 7, 6, 5 and 5, respectively  and the tryptophan residues in BSA, the overlap of the UV/Vis absorption spectra of naringin and naringin palmitate with the fluorescence emission spectrums of BSA at 288 K were studied . The speectively .According to Föster’s non-radiative energy transfer theory, there are main influence factors that influence the efficiency of energy transfer, including the fluorescence production of the donor, the overlap between the fluorescence emission spectrum of the donor and the UV/Vis absorbance spectrum of the acceptor, and the distance between the donor and the acceptor, which is approached and lower than 8 nm SVK of BSA and the warfarin-BSA and ibuprofen-BSA systems were calculated from the slopes of three plots  and just partially overlapped site II (sub-domain IIIA) of BSA and that naringin palmitate was partially located within site I (sub-domain IIA) of the BSA.D-value (Δλ) between the excitation and emission wavelength were stabilized at 15 and 60 nm λ = 15 nm and Δλ = 60 nm. A small red shift (from 288 to 290 nm) of tyrosine residues fluorescence  of naringin palmitate-BSA did not experience a significant shift when the λmax of naringin-BSA shifted slightly towards higher wavelength regions (from 278 nm to 282 nm). Valeur et al. pointed out that the change in λmax indicated a change in polarity around the tryptophan residue, which would change the peptide strand of BSA molecules and the hydrophobicity To reconfirm the structural change of BSA by adding various amounts of naringin and naringin palmitate, the UV/Vis absorbance spectra of BSA were measured, which indicated the change in hydrophobicity and the complex formation −1  and 1548–1560 cm−1  −1 range of the amide I band was attributed to the α-helix −1, respectively. The peak position of amide I moved from 1654 cm−1 to 1656 cm−1, and for amide II, it moved from 1548 cm−1 to 1552 cm−1, upon binding with naringin (−1 to 1655 cm−1, and for amide II, it moved from 1548 cm−1 to 1551 cm−1, upon complexation with naringin palmitate (FT-IR spectroscopy was used  to obtainaringin . The peaalmitate . These rTo clarify the specific changes in the BSA α-helix structure after binding with naringin/naringin palmitate, the far-UV CD spectra of BSA were studied in the presence of different amounts of naringin/naringin palmitate The MRE at 208 nm was regarded as an indicator of α-helix structure in proteins In order to investigate the interactions between substrates and BSA, both rigid and flexible docking studies were performed by AutoDock 4.2 On the other hand, the most likely binding conformation for naringin palmitate is significantly different from naringin in terms of flexible docking binding . NaringiThe conformations that were obtained through rigid and flexible docking studies were compared in The interaction between naringin palmitate and BSA has been studied in great depth using fluorescence quenching, UV absorption spectroscopy, FT-IR, CD spectroscopy, site marker experiments and molecular modelling methods. This work demonstrates that naringin palmitate can bind to BSA and that the addition of naringin palmitate to the buffer solution of protein changes the conformation of BSA, which is substantiated by the CD and FT-IR spectra data. The quenching mechanism of BSA fluorescence by naringin palmitate is static, which is similar to naringin. The temperature affects the stability of the naringin palmitate-BSA complex, and the naringin palmitate and BSA compound is more sensitive to high temperature than naringin-BSA. Displacement experiments and molecular modelling studies suggest that naringin palmitate only partially interacts with site I of the BSA, and the main interaction between them is van der Waals force and hydrogen bonding formation. The distance between naringin palmitate and the BSA binding site is larger than that of naringin, so the naringin palmitate-BSA is less stable than naringin-BSA. In summary, naringin palmitate can bind to BSA and be released more quickly from BSA than naringin, which quickly reaches the maximum concentration. Naringin palmitate can also be transported by proteins and removed easily from the body, and when naringin palmitate is used in a mammal body, it will be effective in improving short-term clinical treatments.