Alzheimer's disease, a common dementia of the elder, is characterized by accumulation of protein amyloid deposits in the brain. Immunization to prevent this accumulation has been proposed as a therapeutic possibility, although adverse inflammatory reactions in human trials indicate the need for novel vaccination strategies.Here vaccination with novel amyloid peptide immunogens was assessed in a transgenic mouse model displaying age-related accumulation of fibrillar plaques.Immunization with any conformation of the amyloid peptide initiated at 12 months of age (at which time fibrillar amyloid has just begun to accumulate) showed significant decrease in total and fibrillar amyloid deposits and in glial reactivity relative to control transgenic animals. In contrast, there was no significant decrease in amyloid deposition or glial activation in mice in which vaccination was initiated at 16 months of age, despite the presence of similar levels anti-Aβ antibodies in young and old animals vaccinated with a given immunogen. Interestingly, immunization with an oligomeric conformation of Aβ was equally as effective as other amyloid peptides at reducing plaque accumulation. However, the antibodies generated by immunization with the oligomeric conformation of Aβ have more limited epitope reactivity than those generated by fAβ, and the microglial response was significantly less robust.These results suggest that a more specific immunogen such as oligomeric Aβ can be designed that achieves the goal of depleting amyloid while reducing potential detrimental inflammatory reactions. In addition, the data show that active immunization of older Tg2576 mice with any amyloid conformation is not as efficient at reducing amyloid accumulation and related pathology as immunization of younger mice, and that serum anti-amyloid antibody levels are not quantitatively related to reduced amyloid-associated pathology. Alzheimer's disease (AD) is an age-related common dementia or loss of cognitive abilities. Neuronal loss, neurofibrillar tangles and senile plaques, abnormal protein deposits which include cleavage products of the amyloid precursor protein (amyloid β peptides (Aβ)) are pathologic characteristics of the disease. While the mechanism of this neurodegeneration remains to be defined, substantial evidence implicating a significant role for the Aβ peptide (40–42 amino acids) has been reported . As aSeveral studies in mouse models have shown that passive immunization, in these cases intracranial or peripheral injection of anti-Aβ antibodies, resulted in relatively rapid clearance of significant amounts of Aβ immunoreactivity, both extracellular deposits as well as intraneuronal Aβ accumulation -15. Furtin vitro that can be reversed by addition of anti-oligomeric antibody [Recent reports have defined an oligomeric conformation of the Aβ structure that alters LTP activity ,22 and iantibody ,24. Sincantibody -27, we tantibody . In addiThe experiments described here were also designed to assess the effect of immunization of animals at an advanced age/stage of pathology on the mitigation of amyloid associated neuropathology in a mouse overexpressing human mutant amyloid precursor protein, and to determine whether differences in complement deposition could be detected on plaques resistant to clearance. Our results, in addition to identifying a novel candidate immunogen, demonstrate that while the level of measured serum antibodies are similar or only slightly different in animals immunized with a given immunogen at different ages, a decrease in the accumulation of both fibrillar and diffuse amyloid plaques occurs only when mice are immunized at early stages of the disease (12–16 months of age). The level of C3 activation fragments associated with plaques was also reduced in animals immunized with any amyloid immunogen, correlating with reduced fibrillar plaque burden. Finally, a new, automated, computer assisted method of quantification of immunoreactivity is described and shown to correlate well with conventional image analysis.2O in a siliconized Eppendorf tube. After 10–20 min incubation at room temperature, the samples were centrifuged for 15 min. at 14,000 × G and the supernatant fraction (pH 2.8–3.5) was transferred to a new siliconized tube and subjected to a gentle stream of N2 for 5–10 min to evaporate the HFIP. The samples were then stirred at 500 RPM using a Teflon coated micro stir bar for 24–48 hr at 22°C. Oligomers were validated by atomic force microscopy (AFM), electron microscopy (EM) and size exclusion chromatography (SEC) as described [1–33) containing both the native B and T cell epitopes of Aβ was synthesized  to increase the response to Aβ. Peptides were resuspended in sterile PBS at 2 mg/ml, vortexed and stored at -70°C.Lyophilized Aβ1–42 peptides were resuspended in 50% acetonitrile in water and re-lyophilized. Soluble oligomers were prepared by dissolving 1.0 mg of peptide in 400 μL hexafluoroisopropanol (HFIP) for 10–20 min at room temperature. 100 μl of the resulting seedless solution was added to 900 μl MilliQ Hescribed . Fibrilsescribed , and sto1–33 peptides were emulsified 1:1 (v/v) with complete Freund's adjuvant containing 4 mg/ml Mycobacterium tuberculosis  [1–33 500 ng of pertussis toxin (PTX)  in 200 ul PBS was injected IP, followed by a second injection 24 hours later [Tg (HuAPP695.K670N-M671L)2576 mice from K. Hsiao  and non-ity, Mo) . Subsequrs later . Immuniz3) at 4°C until use. Fixed brain tissue was sectioned (40 um) with a vibratome, and coronal sections were collected in PBS (containing 0.02% sodium azide), and stored at 4°C prior to staining.Mice were deeply anesthetized with an overdose of pentobarbital , blood collected by cardiac puncture, and then animals perfused transcardially with cold phosphate-buffered saline (PBS). After dissection, brain tissue was fixed overnight with 4% paraformaldehyde in PBS, pH 7.4 at 4°C. Thereafter, fixed tissue was stored in PBS/0.02% sodium azide  at room temperature. Sections were then incubated with primary antibodies (Table Immunohistochemistry (IHC) was performed on free-floating brain sections. To stain for Aβ plaques, sections were immersed in 50% formic acid for 5 min. Endogenous peroxidase in tissue was blocked by treating with 3% HAssays were repeated at least twice, with n = 4–7 animals per group per age per marker as noted in legends and text. Quantitative comparisons were performed on sections processed at the same time. Single ANOVA statistical analysis was used to assess the significance of the differences in plaque area, glial and C3 activation products reactivity among the animals groups.Immunostaining was observed under a Zeiss Axiovert-200 inverted microscope  and images acquired with a Zeiss Axiocam high-resolution digital color camera (1300 × 1030 pixel) using Axiovision 3.1 software. Digital images were analyzed using KS300 analysis program (Zeiss). Percentage of immunostained area  was determined for all the markers studied by averaging % Field Area of several images per section that cover most, or all, of the region of study. 2 of tissue scored and is used as an approximation of the number of plaque-like structures per mm2, and secondly, the ratio (as a percent) of the total area of the Aβ-positive objects to the area of tissue scored. It should be noted that, since the numerator of this second ratio contains only the area enclosed within the boundaries of the identified objects and does not incorporate small particles of Aβ-immunoreactivity that are excluded by the size filter (i.e. <12 microns effective diameter), this measure is distinct from the area ratio described in the previous section and will thus be denoted as the Aβ-Positive Object Area Ratio.A second method of quantification developed for the ACIS image analysis system  was utilized to analyze the 6E10 immunoreactivity. Images were acquired automatically. Cortical and hippocampal regions appropriate for analysis were selected and automatically scored using an algorithm that identifies objects based on user-configurable parameters. Object identification was paired with a watershed segmentation algorithm to facilitate separation of touching and overlapping deposits. In this manner large deposits that form contiguous bands of Aβ were separated into individual objects. Because Aβ deposits in these animals can vary markedly in size and shape, the identification of Aβ-positive objects utilized a broad size filter  and did not employ rigorous morphometric filters. Data collected for each Region of Interest (ROI) were the area of tissue scored (area of the ROI), the number of Aβ-positive objects identified and the total area of the Aβ-positive objects. These parameters allowed calculation of two different measures of amyloid load: the Aβ-Positive Object Density, which is simply the number of objects per mmall ROIs. This analysis was performed blinded to results from prior conventional quantification as described above.Comparisons among experimental groups were based on single values per animal for Aβ-Positive Object Density and Aβ-Positive Object Area Ratio. These were calculated by determining the sum of the numbers of Aβ-positive objects (or the sum of the areas of the Aβ positive objects) for the entire section and dividing by the sum of the areas of ELISA assays were performed as previously described . Briefly1–33 or adjuvant alone. Representative photomicrographs presented in Figure 1–33 was used as an immunogen, fibrillar plaque accumulation was decreased by 53% relative to adjuvant control  by 52% and 44% relative to adjuvant and untreated control respectively .To assess total amyloid deposits in the APP transgenic mice after the 4 month regime of immunization, the human Aβ specific monoclonal antibody, 6E10, was used as described in Material and Methods. Figure 1–33 immunogen, resulted in significant changes in Aβ deposition, with 2- and 3-fold reductions in both plaque number (Aβ-Positive Object Density) and plaque area (Aβ-Positive Object Area Ratio) relative to the respective adjuvant controls (p < 0.05 by two-tailed t-test). In particular, the extent of the reduction in the novel measure of Object Density by Aβ, oligo or the MAPAβ1–33 immunized groups was essentially identical compared to the adjuvant controls, . The significant decreases in plaque area in Aβ, oligo or the MAPAβ1–33 immunized groups obtained by this method were 1.96, 2.39, and 2.72-fold. These quantitative results correlated well with conventional image analysis of the same specimens , providing additional validation to the conclusion that immunization with three different forms of the amyloid peptide result in a similar decrease in amyloid plaque accumulation in this murine model when immunized during a period of rapid amyloid deposition.To further validate the quantification of these immunohistochemical results, sections were also analyzed using an automated digital imaging system . A digital image was acquired for the entirety of each section at a resolution of one pixel per micron. All cortical and hippocampal tissues were then analyzed by object identification and feature extraction algorithms. Since analysis of over 50 sections containing both cortex and hippocampus demonstrated that immunoreactivity was higher in the cortex than in the hippocampus, only sections containing cortex and hippocampus were used in these treatment comparisons. Both oligomeric and fibrillar Aβ conformations, as well as the MAPAβCharacteristic of fibrillar amyloid plaques in both human AD brain and in mouse models of amyloid accumulation is robust association of activated astrocytes, which can be envisioned by the astrocyte cell marker GFAP. GFAP staining of B6/SJL control animals was negligible relative to the APP transgenic animals, whether untreated, immunized with adjuvant only or any Aβ immunogen (data not shown). However, while prominent plaque associated GFAP staining was seen in both untreated and adjuvant only transgenic mouse brain, the mean GFAP % field area after the 4 month immunization scheme described in M & M with oligo Aβ or fAβ was 42 and 41% respectively of CFA control, ie a decrease of 58% and 59% Figure  and 4B. 1–33 also showed significantly less CD45   Figure  and MAC-1–33 immunized animals).The identical immunization scheme was performed with older Tg2576 and wild type (B6/SJL) animals. Immunization was initiated at 16 months of age, a time at which there was robust total and fibrillar amyloid deposits . Furthermore, as would be expected after 4 months of immunization, no appreciable IgM anti oligomeric Aβ or anti fibrillar Aβ was detected (using an ELISA specific for IgM detection). Only very low levels of IgM anti soluble Aβ was observed in sera from animals immunized with fibrillar Aβ.Levels of anti-amyloid antibody induced in all immunized animals differed to some extent among the immunogen and the age of animals at the time of immunization, although all animals responded to immunization. A most notable observation was that immunization of both transgenic and wild type mice with the oligomeric conformation of Aβ resulted in measurable antibody to the oligomeric form of Aβ Table , with trSA Table , but als1–33 animals all stained cortical plaques (data not shown).To determine if the antibodies generated with each immunogen were able to bind amyloid plaques in the APP transgenic mice, dilutions of sera from each group of animals immunized at 12–16 months and animals immunized with fAβ at 16–20 months were screened for the ability to bind to amyloid deposits in brain sections from an unimmunized 16 month APP mouse. Consistent with the ELISA data above, antisera from oligo immunized animals did not stain plaques, whereas antisera from animals immunized at 12–16 months and 16–20 months with fAβ and sera from MAPAβ1–33(n = 4) at 12–16 months that were tested were negative for plaque-associated mouse IgG. Sections from each animal were tested 2–4 times. Thus, only animals immunized with fAβ at 12–16 months showed any plaque-like IgG deposition in brain tissue, even though the titer of anti fibrils and anti-soluble Aβ, were generally higher in the MAPAβ1–33 immunized animals.As an indication of whether any induced antibody to β-amyloid had access to the brain, sections from brain of immunized animals were tested for immunoreactivity to mouse IgG using two approaches: direct staining with biotinylated anti mouse IgG Figure  and indiComplement activation products associated with plaques have been detected in murine models of AD ,35. Sinc1–33, induced antibodies to oligomeric, fibrillar and soluble Aβ. Thus, one could speculate that the lower microglia activation is the result of this restricted induction and that this feature may lead to a lower potential for autoimmune responses and inflammatory responses. Further delineation of the basis for the apparently lower microglial activation is, however, necessary to substantiate this hypothesis. Since we did not stain these sections for T cells, it is unknown if the Oligo Aβ immunogen elicited a strong T cell mediated immune response beyond the induction of anti-oligomeric antibody. While this is a concern since a fraction of patients in the human amyloid immunization trial (AN1792) developed meningoencephalitis [The data presented here is the first investigation of the effect of active immunization with amyloid beta peptide in a specific oligomeric conformation in a murine mouse model of Alzheimer's Disease. Immunization with this form of the peptide was as efficient at reducing amyloid deposition in Tg2576 animals as either fibrillar Aβ or a multiple Aβ antigen peptide when immunization was initiated at 12 mos. Interestingly, there was significantly less (p < 0.05) activated microglia in the animals immunized with the oligomeric form of Aβ than in those immunized with fAβ. In addition, the antibody response induced by immunization with oligomeric Aβ had a much more restricted epitope response, generating antibodies to oligomeric Aβ, but not to soluble or fibrillar Aβ, similarly to that observed in vaccination of rabbits with the oligomeric conformation of Aβ . The othrial AN172 developphalitis , any redIn contrast, no significant decrease was seen in any pathological marker measured in mice vaccinated with any peptide form when vaccination was initiated at 16 months, a time at which there are high levels of accumulated plaques. The lack of efficacy in reduction of plaque associated pathology by immunization of these older mice, similar to that reported by Das, et al , was not1–33 resulted in similar 2.63- and 2.72-fold reductions in Aβ-Positive Object Density (i.e. plaque number) and Aβ-Positive Object Area Ratio (i.e. plaque area), respectively. In contrast, fibrillar Aβ immunization affected these parameters somewhat differently, resulting in a 2.73-fold reduction in Aβ-Positive Object Density but a lower (though still significant) 1.96-fold reduction in Aβ-Positive Object Area Ratio. This raises the intriguing possibility that different immunization paradigms might effect change in amyloid burden preferentially through alteration in either plaque number or in plaque size, although additional study will be required to rigorously test such an assertion.In this study we used a second, novel method for quantification of immunohistochemical detection, specifically of amyloid deposits. Although image analysis can bring accuracy and objectivity to IHC, certain drawbacks have historically limited its use. Not only are the processes of image acquisition and data management labor-intensive, but the mechanics of analysis itself can introduce some subjectivity. Automated digital imaging systems, such as the one tested here, have been developed to address such drawbacks. Results from analysis of amyloid burden using such an automated platform correlated tightly with those obtained with an accepted more traditional image analysis approach. Thus, this method is shown to accurately assess amyloid burden in transgenic mouse models of AD. In addition, the use of object-based analysis provides data not obtainable through simple computation of % area positive. For example, immunization with MAPAβ1–33 contained levels of anti-fibrillar Aβ similar to the fAβ immunized animals, but no IgG was detected on amyloid plaques even in the younger set (immunization initiated at 12 m of age) of animals (n = 6) in which reduction of plaques and associated pathology was comparable to fAβ immunized animals. These data suggest that there may be differences in access of the antibody to brain tissue via blood brain barrier or IgG-transport systems in mice immunized with fAβ . Multipcificity . Howeverlleagues .Finally, the amount of C3 activation fragments deposited on the plaques in all animals was largely correlated with the amount of thioflavine staining detected in each animal. While this would be expected since fibrillar amyloid has been shown to activate complement -43, it i40 from the brain supporting the role of LRP-1 as a transporter of Aβ peptide out of the brain [The mechanism by which immunization schemes decrease neuropathology and prevent or reverse behavior deficits has not yet been defined. A decrease in amyloid deposition as a result of active immunization was seen in APP overexpressing transgenic animals that are FcR gamma chain deficient , suggesthe brain . Moreovehe brain . InteresAnother possible mechanism proposed is that antibodies inhibit fibrillization of amyloid and/or promote depolymerization of fibrils (reviewed in ). Still A hypothesis consistent with the observations in both human AD and transgenic mouse models of AD is that there are at least two, likely overlapping, stages contributing to neuronal dysfunction in AD. Early events involving the intracellular accumulation of Aβ peptides  and/or gIn summary, immunization with any amyloid peptide immunogen resulted in significant reduction (45–55%) of plaque pathology and decreased inflammatory microglial cell reactivity (40–65%) only when immunized prior to the massive deposition of large mature plaques. These decreases in pathology did not necessarily correlate with the level of anti Aβ1–42 IgG in sera or with plaque-associated IgG. This first analysis of deposition/accumulation of complement C3 activation within an immunization trial shows association of C3b/iC3b with plaques that correlates with the amount of thioflavine plaques present rather than any association with the antibody response induced. Finally, immunization with oligomeric Aβ1–42 induced a greater decrease in "reactive" microglia relative to immunization with fibrillar Aβ as determined by microglial surface expression of CD45 and MAC-1 antigens. The lower microglial response resulting from immunization with oligomeric Aβ suggests that the goal of depleting plaques can be accomplished while inducing less neuroinflammation, and thus facilitating the application of immunotherapy to treatment and/or prevention of AD in humans.AD, Alzheimer's Disease; BBB, blood brain barrier; CFA, complete Freund's adjuvant; fAβ, fibrillar amyloid peptide 1–42; IFA, incomplete Freund's adjuvant; oligo Aβ, Aβ 1–42 conjugated to colloidal gold; PT, pertussis toxin.Charles Glabe and Rakez Kayed are consultants for Kinexis, Inc. and Scott Webster is an employee at Clarient, Inc. The authors declare that they have no other competing interests.JZ performed all animal treatments, perfusions, immunohistochemistry, image analysis, data analysis and presentation, and guided or performed tissue preparation and serum collection. MF performed immunohistochemistry, image analysis, data analysis and presentation, molecular genetic studies and manuscript preparation; RK prepared immunogens and performed immunoassays, S.W. performed image analysis, data analysis and manuscript preparation; IH performed immunohistochemistry, image analysis, data analysis and presentation and assisted with perfusions and animal care; OY performed immunohistochemistry and image analysis; DC assisted with the design of the study and manuscript preparation; CG contributed to the design of the study and data analysis, and manuscript preparation. AJT developed the design of the study, guided study execution, directed data analysis, interpretation and presentation, and drafted and edited the manuscript. All authors read and approved the final manuscript.


We compared simultaneous measurements of blood lactate concentration ([Lac]) in the right atrium (RA) and in the pulmonary artery (PA). Our aim was to determine if the mixing of right atrial with coronary venous blood, having substantially lower [Lac], results in detectable decreases in [Lac] from the RA to the PA.2 saturation (SO2) and [Lac]. We defined Δ[Lac] as ([Lac]ra - [Lac]pa), ΔSO2 as (SraO2 - SpaO2) and the change in O2 consumption (ΔVO2) as the difference in systemic VO2 calculated using Fick's equation with either SraO2 or SpaO2 in place of mixed venous SO2. Data were compared by paired Student's t-test, Spearman's correlation analysis and by the method of Bland and Altman.A prospective, sequential, observational study was conducted in a medical-surgical intensive care unit. We enrolled 45 critically ill adult individuals of either sex requiring pulmonary artery catheters (PACs) to guide fluid therapy. Immediately following the insertion of the PAC, one paired set of blood samples per patient was drawn in random order from the PAC's proximal and distal ports for measurement of hemoglobin concentration, OraO2 > SpaO2  and [Lac]ra > [Lac]pa . Δ[Lac] correlated with ΔVO2 .We found S2 and [Lac] from the RA to PA support the hypothesis that these gradients are produced by mixing RA with coronary venous blood of lower SO2 and [Lac]. The present study is a preliminary observation of this phenomenon and further work is needed to define the physiological and clinical significance of Δ[Lac].We found decreases in [Lac] from the RA to PA in this sample of critically ill individuals. We conclude that parallel decreases in SO The insertion technique is described elsewhere . Care wa2 saturation was determined from a previously in vivo calibrated pulse oximeter.Immediately after the insertion of the PA catheter, each patient had one set of paired blood samples drawn in rapid succession, and in random order, from the proximal and distal port. We took proximal port blood to be representative of RA blood, whereas distal port blood was considered to be PA blood. The first 2 ml of blood drawn for each sample were discarded to prevent contamination with flushing fluid. Blood samples were drawn with the catheter balloon deflated to avoid contamination of the distal port sample with pulmonary capillary blood. Arterial O2 saturation . We measured cardiac output (CO) by the thermodilution method as the average of three sequential determinations.Blood samples were placed on ice and taken to a central laboratory for measurement of [Lac] , hemoglobin concentration ([Hb]) and O2 delivery (DO2), O2 consumption (VO2), O2 extraction ratio (ERO2), double product  × mean arterial pressure (MAP)) and left ventricular stroke work index (LVSWI) were computed using standard formulae. We defined ΔVO2 as the difference in systemic VO2 calculated with Fick's equation with either SpaO2 or SraO2 in place of the mixed venous SO2 (SvO2); ΔVO2 = Qpa × 13.4 × [Hb] × (SraO2 - SpaO2) ml.min-1.Systemic Ora and [Lac]pa were compared by Spearman's correlation analysis analysis . The metanalysis  was usedWe enrolled 45 patients in the study, including 18 women. The study group was composed of 31 post-operative patients (26 post-cardiac surgery), 11 patients in septic shock from various medical conditions, 2 patients with severe gastrointestinal bleeding and 1 patient in congestive heart failure. Demographic and hemodynamic parameters for the group are listed in Table 2 and lactate concentrations for RA and PA blood samples are shown in Table raO2 was greater than SpaO2 (p < 0.001), with ΔSO2 = 5.2 ± 4.8%. [Lac]ra was greater than [Lac]pa (p < 0.001), with Δ[Lac] = 0.2 ± 0.2 mmol.l-1.The mean SOra and [Lac]pa. There was a bias towards greater [Lac]ra of 0.2 mmol.l-1 (p < 0.001) with a 95% confidence interval for the population of -0.15 to 0.56 mmol.l-1. There was no discernable relationship between [Lac]ra and Δ[Lac] , indicating that Δ[Lac] was not a concentration dependent phenomenon. Moreover, we found no significant relationships between [Lac]ra and SraO2 or between [Lac]pa and SpaO2.Shown in Fig. 2  with a standard error of the estimate of 0.15 mmol.l-1  drawn from 12 critically ill patients [Only one other study in the literature has compared central venous [Lac] to [Lac]-1. Even assuming a worst case scenario of a systematic instrument bias of -0.1 mmol.l-1, the difference in [Lac] between RA and PA would have remained statistically significant.We used a standard clinical laboratory instrument to measure [Lac] having a 95% precision of ± 0.1 mmol.lThe declining [Lac] gradient from RA to PA is likely the result of mixing RA blood with blood of lower [Lac] emanating from the coronary venous system. Lactate oxidation accounts for 10% to 20% of total myocardial aerobic energy production , a propopa. A factual resolution of this question can only be achieved by direct measurement of [Lac] from IVC to PA.Since RA blood is the mixture of superior vena cava and inferior vena cava (IVC) blood, the possibility exists that these blood streams had not thoroughly mixed at the proximal PAC sampling port. In this case, one could expect further mixing to occur between IVC and RA blood while flowing into the pulmonary artery. Our results do not support this hypothesis. Direct measurements in humans show that IVC blood has the highest [Lac] of any major vein  and furtra < [Lac]pa. These patients had no distinguishing features to help us differentiate them from others in the group. It is possible that accidental mislabeling of the samples may have accounted for a negative Δ[Lac] but we think it unlikely, given the care taken with the labeling and measuring of the samples. Another possibility is that these individuals experienced myocardial ischemia, a condition associated with an upsurge in glucose metabolism and net lactate release by the heart [ra < [Lac]pa.Only three individuals in our group had [Lac]he heart -19. Myoc2 consumption (MVO2) and myocardial lactate uptake, reflecting the O2 cost of lactate utilization by the heart  (Fig. 2 and myocardial lactate uptake. This finding suggests that Δ[Lac] also may be related, in a yet to be established fashion, to MVO2.Others have noted a linear relationship between myocardial Ohe heart . We did 2 and [Lac] from RA to PA support the hypothesis that these gradients are produced by mixing RA with coronary venous blood of lower SO2 and [Lac]. The present study is a preliminary observation of this phenomenon and further work is needed to define the physiological and clinical significance of Δ[Lac].We found decreases in [Lac] from RA to PA in this sample of critically ill individuals. We conclude that parallel decreases in SO• 	Oxygen and lactate concentrations are lower in PA blood than in RA blood.• 	The oxygen and lactate concentration gradients from RA to PA are likely the result of mixing atrial with coronary venous blood.• 	The possibility exists that these concentration gradients may reflect changes in myocardial energy requirements.2 = systemic O2 delivery; DP = double product; ERO2 = oxygen extraction ratio; [Hb] = hemoglobin concentration; HR = heart rate; IVC = inferior vena cava; Δ[Lac] = lactate concentration gradient from right atrium to pulmonary artery; [Lac] = blood lactate concentration; LVSWI = left ventricular stroke work index; MAP = mean arterial pressure; MPP = mean pulmonary pressure; MVO2 = myocardial O2 consumption; PA = pulmonary artery; PAC = pulmonary artery catheter; PAOP = pulmonary artery occlusion pressure; RA = right atrium; SO2 = O2 saturation; ΔSO2 = O2 saturation gradient from right atrium to pulmonary artery; SVRI = systemic vascular resistance index; VO2 = O2 consumption.CV = coronary venous; CVP = central venous pressure; DOThe authors declare that they have no competing interests.GG conceived the study, participated in its design, performed statistical analysis and drafted the manuscript. LSC and HZ participated in the design of the study, collected data and helped to draft the manuscript. MGS and NMK conducted the study, collected data and helped to draft the manuscript. All authors read and approved the final manuscript.


When reporting incidence rate estimates for relatively rare health conditions, associated case counts are often assumed to follow a Poisson distribution. Case counts obtained from large-scale electronic surveillance systems are often inflated by the presence of false positives, however, and adjusted case counts based on the results of a validation sample will have variances which are hyper-Poisson. This paper presents a simple method for constructing interval estimates for incidence rates based on case counts that are adjusted downward using an estimate of the predictive value positive of the surveillance case definition. Large-scale surveillance for selected medical or health conditions often relies on electronic data sources which provide comprehensive coverage of a given population. For example, the Centers for Disease Control and Prevention conduct surveillance of brain injuries involving hospitalization or death, based on electronic hospital discharge and vital statistics data received from twelve to fifteen states each year . To idenAs with most surveillance methods, an operational case definition as described above may admit some records that do not represent true cases under a strict clinical definition  and may also fail to capture some records representing true cases . The customary terms reflecting these aspects of an operational case definition are predictive value positive (PVP) and sensitivity, defined in the present context as the conditional probabilities :PVP = Pr{case meets clinical definition | case detected under operational definition};sensitivity = Pr{case detected under operational definition | case meets clinical definition}.Depending on the extent to which false positives and/or false negatives are believed to influence the surveillance process, it may be appropriate to use estimates of PVP and/or sensitivity to adjust incidence rate estimates accordingly. It is not generally possible to assess PVP or sensitivity using electronic surveillance data alone. The most direct approach to obtaining the additional data required for estimation of PVP involves manual review of medical records for a random sample of provisional cases identified via the operational case definition. Obtaining the additional data necessary for estimation of sensitivity may be more labor-intensive, particularly when considering an uncommon condition. Without additional "markers"  to narrow the scope of review, it may be necessary to select a very large sample of general medical records in order to identify enough true cases to support a stable estimate of sensitivity.The methodology described in this paper is oriented to surveillance of relatively rare health conditions. Because validation data quantifying the influence of false positives will typically be easier to obtain than data quantifying the influence of false negatives in this setting, the development concentrates on incidence rate estimates reflecting adjustments for PVP. This emphasis is not intended to diminish the potential influence of false negatives; rather, it reflects the logistical difficulties associated with obtaining data on false negatives as part of ongoing surveillance. If there is sufficient doubt surrounding the sensitivity of case ascertainment for any particular surveillance process, the proposed methodology should be applied with due caution.For a given surveillance period, it is assumed that case confirmation data are available for a random sample (selected without replacement) of provisional cases. Data obtained through such validation efforts allow estimation of PVP as well as adjustments to case counts to eliminate the bias due to false positives. To illustrate, suppose that for a set period  of observation:N = size of the at-risk population covered by the surveillance system;M = count of provisional cases detected under the operational case definition;T = count of true cases (unknown) among the provisional cases;MF = count of false positive cases (unknown) among the provisional cases = M - MT;MS = number of provisional cases sampled for case confirmation;T = count of confirmed true cases among those sampled;CF = count of cases determined to be false positives among those sampled = S - CT.CThe usual estimate of PVP is given by : = CT/S = CT/(CT + CF). is definable only when M > 0  a reasonable estimate of the population of true cases which eliminates the false positive bias is:Noting that T could be determined. When reporting the corresponding incidence rate R = MT/N one might also make use of the variance estimate , based on the assumption that MT represents one observation from a Poisson process  = τ        (2)E = M·PVP for M ≥ 0. Applying principles of conditional expectation  = E[E[|M]] = E[M·PVP] = λ·PVP.E[) it is convenient to employ the following variance decomposition [To determine Var(position :) = E[Var(|M)] + Var(E[|M]).Var(|M] = M·PVP it follows that Var(E[|M]) = λ·PVP2. Evaluation of the first component of variance is more complicated. Defining:Since E ≤ E[M/f] = λ/f. Given fixed f the upper bound is a good approximation provided that λ is sufficiently large, so that E[g(M)] ≌ λ/f and E[Var(|M)] ≌ PVP·(1-PVP)·λ/f. Combining variance components and simplifying results in:The task is thus reduced to determining E[g(M)]. When s(M) = ) across a range of values for PVP, λ, and f shows that for f ≥ 0.01 and f·λ ≥ 50, the relative error of (A.2) is less than 0.01.Numerical calculation of Var(The following is proposed as an estimator of the right-hand side of (A.2):Defining:it follows from the treatment in Appendix A that the expected value of the variance estimator (B.1) conditioned on M is: it follows that:Then, since  it holds that h(M) ≤ 1/f and hence that E[h(M)] ≤ 1/f. Given fixed f the upper bound is a good approximation provided that λ is sufficiently large. Substituting 1/f in place of E[h(M)] results in:When s(M) = Algebraic simplification results in:As f·λ becomes large, approximation (A.2) results.The author(s) declare that they have no competing interests.


Synaptic dysfunction due to the disrupting binding of amyloid beta (Aβ) and tau oligomers is one of the earliest impairments in Alzheimer’s Disease (AD), driving initial cognitive deficits and clinical manifestation. Consequently, there is ample consensus that preventing early synaptic dysfunction would be an effective therapeutic strategy for AD. With this goal in mind, we investigated the effect of a treatment of mice with near infrared (NIR) light on synaptic vulnerability to Aβ oligomers. We found that Aβ oligomer binding to CNS synaptosomes isolated from wild type (wt) mice treated with NIR light was significantly reduced and the resulting suppression of long term potentiation (LTP) by Aβ oligomers was prevented. Similarly, APP transgenic mice treated with NIR showed a significant reduction of endogenous Aβ at CNS synapses. We further found that these phenomena were accompanied by increased synaptic mitochondrial membrane potential in both wt and Tg2576 mice. This study provides evidence that NIR light can effectively reduce synaptic vulnerability to damaging Aβ oligomers, thus furthering NIR light therapy as a viable treatment for AD. Furthermore, we recently reported a group of individuals referred to as Non- Demented with Alzheimer’s Neuropathology (NDAN) that provides evidence supporting the correlation between the presence of Aβ oligomers at the synapses and the retention of cognitive function. NDAN individuals maintain their cognitive integrity despite the presence of Aβ plaques and neurofibrillary tangles at an extent comparable to demented AD patients. By comparing these individuals to AD patients, one can infer properties that may protect these individuals from the cognitive dysfunction normally associated with AD pathology. We showed that while NDAN individuals displayed similar levels of soluble Aβ oligomers throughout their CNS, contrary to demented AD patients, they had synapses that were devoid of Aβ oligomers7. This suggests the possibility that synapses of NDAN subjects are resistant to Aβ oligomers and illustrates that absence of Aβ at synapse is a key event associated with preservation of cognitive integrity. Taken together this evidence suggests that eliciting a protective mechanism resulting in synaptic resilience to binding of Aβ oligomers similar to NDAN individuals would be the most effective protection against Aβ oligomer driven toxic synaptic dysfunction. With this goal in mind, in the present work we investigated the effect of a treatment with near infrared light  in increasing synaptic resilience to Aβ oligomers. NIR light treatment administered in a noninvasive transcranial application has been suggested to photostimulate the mitochondrial chromophore, cytochrome c oxidase, resulting in increased ATP formation11. Mitochondrial dysfunction is a pathological event occurring in early stages of AD. Much evidence suggests dysfunctional mitochondria events such as increased reactive oxygen species (ROS) production13, cytochrome c deficiencies15, and mutated mitochondrial DNA17 contributes to the exacerbation of AD pathology19. Notably, studies have described the intimate relationship between reduced Aβ oligomer binding at synapses and increased mitochondrial function20. Thus, it is not unreasonable to hypothesize that NIR light treatment could effectively promote synaptic resilience to Aβ oligomer binding. To test this hypothesis, we investigated the ability of NIR light to reduce synaptic susceptibility to Aβ oligomer binding and, as a result, increase synapse function. We utilized wild type (wt) mice to determine the impact of NIR light treatment on the binding of Aβ oligomers to isolated synaptosomes and long term potentiation (LTP) in the hippocampus of these NIR light-treated wt mice in the presence and absence of Aβ oligomers. To determine light-driven changes in the synaptic presence of endogenous Aβ oligomers, we also investigated the Aβ1–42 load at isolated synapses of NIR light-treated Tg2576 mice that overexpress human amyloid precursor protein (APP) and progressively accumulate Aβ in their CNS24. We further investigated synaptic mitochondria in wt and Tg2576 mice after NIR light treatment. We found that NIR light treatment reduced ex vivo Aβ oligomer synaptic binding in wild type mice that was paralleled by a retention of long term potentiation induction. We further found that after NIR light treatment, the levels of Aβ1–42 at the synapses was significantly reduced in 6-month-old Tg2576 mice. These changes were in conjunction with a retention and increase in the synaptic mitochondria health after NIR light treatment in both wt and Tg2576 mouse models.Alzheimer’s disease (AD) is the most common and severe age-associated neurodegenerative disorder for which there is currently no effective therapeutic intervention. One of the key events contributing to the development of the cognitive decline that marks the clinical profile of AD is the selective targeting and disruption of the synapses by small, soluble amyloid beta (Aβ) oligomers, the most toxic form of the Aβ protein. Aβ oligomers binding to synapses have been shown to induce many morphological and physiological changes that collectively lead to the loss of cognitive integrity including retraction of synaptic spinesex vivo Aβ oligomer binding on isolated synaptosomes from wild type mice receiving NIR light treatment. The NIR light treatment consisted of a 90 second exposure  hippocampal and cortical synaptosomal fractions from mice treated with NIR light had decreased Aβ binding at the concentrations of Aβ oligomers used  was reduced in the group receiving NIR light treatment as compared to the sham-treated group in both the hippocampus  and the cortex . On the other hand, no changes in the affinity (Kd) of Aβ binding were observed between groups for either the cortex or hippocampus . The smaller Bmax along with unchanged Kd indicates that there was a reduction in the number of binding sites at the synapses in the group that received NIR light treatment.To investigate the effects of NIR light on synaptic sensitivity to Aβ, we studied the ure Fig.  administure Fig. . The parsed Fig. . We furtex vivo Aβ oligomer binding at a single concentration (5 μM) on synaptosomes prepared from each individual wt mouse  to further demonstrate changes in binding properties due to NIR light treatment. Consistent with the results described above, we found that in the parieto-occiptial cortex, frontal cortex, and hippocampus there was a significant reduction of Aβ1–42 positive synaptosomes of about 7%, 16%, and 6%, respectively, in the samples prepared from NIR light treated mice as compared to the control, sham-treated animals  and the mice sacrificed one month later. Levels of Aβ in the synaptosomal fractions and total homogenates from the frontal cortex, hippocampus, parieto-occipital cortex, and cerebellum were analyzed by a specific ELISA. As shown in Fig. In order to determine the effect of NIR light treatment in protecting synapses from endogenous Aβ oligomers 26. As shown in Fig. In order to determine whether the reduced susceptibility of synapses to Aβ oligomers would translate into functional protection, we utilized wild type mice to determine if NIR light treatment rescues the synaptic impairment of LTP that normally occurs in response to exposure to Aβ oligomers28 and reported that preserving synaptic mitochondria efficiency protects synapses from Aβ oligomers28. We first analyzed by flow cytometry synaptosomes from wild type and Tg2576 mice treated or not with NIR light that had been labeled with MitoTracker, a mitochondrion-specific fluorescent dye whose stain intensity is directly proportional to the number of mitochondria29. We found that there was a decreased number of synaptic mitochondria in Tg2576 mice as compared to age matched wild type mice . The decreased Bmax value in the NIR light treated groups with no change in the affinity (Kd) suggests that after NIR light treatment there is a reduction in Aβ oligomer docking sites at the synapse, providing compelling evidence that a secondary mechanism of action of the NIR light treatment is contributing to the increased resilience to the toxic protein. By gating our observation to selected particle sizes, flow cytometry further allowed us to exclude nonspecific binding to nonsynaptosomal particles.Synaptic function is an essential element in the maintenance and preservation of cognition24 to determine whether an in vivo reduction of endogenous Aβ at the synapses could be observed after NIR light treatment. Previous research has shown a reduction of the Aβ plaque load after NIR light treatment in the cortex and hippocampus of APP/preselinin 1 (PS1) double transgenic mice38. However, this previous study focused on overall Aβ levels in plaques and did not address the effect of NIR light on synapse vulnerability to the toxic Aβ oligomers, as we have determined here. To provide a complete understanding of the impact NIR light has on synaptic vulnerability to Aβ, in our study Tg2576 mice were treated with NIR light and the Aβ levels were measured in both total brain protein extracts as well as synaptosomal fractions. We employed Tg2576 mice at age of 6 months when ample Aβ load, mainly in the form of oligomers, is seen and cognitive defects are prevalent, but plaques formed by large Aβ fibrils deposition are not yet present24. Therefore, Tg2576 mice at 6 months of age are an ideal model to study specifically the impact of Aβ oligomers. Our results showed a significant decrease of Aβ load in the synaptosomes from all four brain areas tested, i.e. parieto-occipital cortex, hippocampus, frontal cortex and cerebellum. However, the total brain tissue homogenates showed a significant decrease of Aβ only in the parieto-occipital cortex. The other three brain regions showed a trend of decreased levels, however, they reduction was not statistically significant. The differential impact of the NIR light among the brain regions may be attributed to higher energy exposure to superior structures or higher sensitivity to NIR light of the parietal-occipital cortex, although further experiments are needed to support either (or both) of these possibilities. The differences in reduction between the total homogenate and synaptosomes suggest that NIR light initiates a mechanism that targets specifically the synapses. Interestingly, this selective reduction of Aβ at synapse induced by the NIR light treatment is similar to what observed in the cognitively intact NDAN individuals. As we have previously described, NDAN individuals have presence of Aβ oligomers in the CNS at levels similar to demented AD patients, however, they have an absence of Aβ oligomers at the synapses, which suggests synaptic resistance to oligomer binding7. The results of our in vivo experiment thus suggest that NIR light treatments may effectively evoke a mechanism of synaptic resilience similar to the natural ability to resist the binding of Aβ oligomers seen in NDAN individuals with preserved cognitive integrity.We utilized the Tg2576 mouse strain that overexpresses human APP and progressively accumulate Aβ oligomers39. A well-established impairment that occurs when Aβ oligomers bind to synapses is reduced magnitude of LTP40 and increased long term depression (LTD)41. In the present study, we aimed to determine if NIR light reversed this observed reduction of LTP expression by Aβ oligomers. Consistent with those previous experiments, we found a significant reduction in the magnitude of LTP in wt mice incubated with 200 nM of Aβ oligomers. However, after NIR light treatment wt mice demonstrated an induction of LTP similar to the control group without Aβ oligomer exposure. This suggests that the reduction of Aβ oligomer binding that we observed following NIR light treatment in the ex vivo studies results in a significant rescue in synapse functionality. The electrophysiology experiment was conducted after treating the wt mice with a condensed treatment protocol. We found that mice receiving the same number of treatments  and the same delivery of total energy as the month-long treatment over a course of 5 days showed similar reductions in the synaptic binding of Aβ oligomers  and synaptic mitochondrial abundance. Consistent with the literature, we found a decrease in the abundance of synaptic mitochondria in Tg2576 mice as compared to age matched wild type mice48 and further observed that after NIR light treatment, this deficiency in the synaptic mitochondria was rescued. On the other hand, wild type mice treated with NIR light did not have an increase in synaptic mitochondria compared to their control treatment counterparts. This suggests that under normal physiological conditions, NIR light does not induce an increase of mitochondria numbers at the synapses. However, the rescued synaptic mitochondria number that is seen in the NIR light-treated Tg2576 synapses suggests that the NIR light-induced reduction of Aβ oligomer binding contributes to the reversal of the depletion of synaptic mitochondria. Nonetheless, given the fact that the beneficial effect of NIR light in promoting synaptic resilience to Aβ oligomers was observed in both wt and Tg2576 mice, synaptic mitochondrial abundance does not appear to be a key event in this action of NIR light. On the other hand, when we examined the membrane potential of the synaptic mitochondria as an indicator of mitochondrial health after NIR light treatment, we found that both wild type and Tg2576 mice showed an increase in synaptic mitochondrial membrane potential after treatment with NIR light. Previous studies have shown a direct relationship between synaptic vulnerability to Aβ oligomer binding and the health of synaptic mitochondria28. Furthermore, synaptic mitochondria are believed to have an integral role in the regulation of LTP induction52. Therefore, our results suggest a mechanism of NIR light action critically involving increasing synaptic mitochondrial membrane potential, consistent with the reported increase in synaptic resilience to Aβ oligomers induced by promotion of synaptic mitochondrial function/health. Taken together these results indicate that NIR light treatment increases synaptic mitochondria health, thus decreasing synaptic susceptibility to Aβ binding and consequent electrophysiological deficits in LTP expression.A possible mechanism that could be contributing to the protection and increased function of synapses seen in our study is increased function of the synaptic mitochondria. The 600–1000 nm wavelength spectrum of the NIR light has been shown to optimally photostimulate cytochrome c oxidase, a key enzyme in the electron transport chain that is believed to result in an increase in ATP productionCollectively, the results of our study provide valuable insight into the mechanisms that underscore the reported mitigation by NIR light of AD pathology and further introduce a previously unappreciated phenomenon of increased synaptic resilience to the disrupting action of Aβ oligomers that can be promoted by NIR light exposure.in vivo Aβ load at the synapses , as well, as the electrophysiological properties after NIR light treatments . C57BL/6 wild-type male and female mice were employed for the ex vivo Aβ binding experiments . Male and female Tg2576 and wild type mice were utilized for synaptic mitochondrial experiments . All experimental protocols involving animals were approved by Institutional Animal Care and Use Committee of the University of Texas Medical Branch. All methods were performed in accordance with the guidelines and regulations of the committee. Animals were housed under USDA standards  at the UTMB vivarium.Male and female transgenic 2576 (Tg2576) mice were utilized to evaluate After the conclusion of the NIR light treatment, the mice were sacrificed by exposure to isoflurane and decapitated. The brains were quickly removed, dissected into major regions; frontal cortex, parieto-occipital cortex, hippocampus and cerebellum and stored at −80 °C until ready for further analysis.2 per treatment. The treatment group received one dose per day, 5 days per week for 4 consecutive weeks. The mice were hand restrained and the light device was held approximately 1 cm from the top of the head. The body of the mouse was covered with aluminum foil to prevent light exposure to the periphery, as illustrated in Fig. 38. The NIR light treatment was condensed to one week (4 treatments per day for 5 days) for wt mice treated for the electrophysiology experiment after it was determined that this condensed time period provided similar reduced binding effects as the 4 week treatment schedule  device . Light energy emitted equated to 4 Joule/cm53.After the final NIR light treatment, the animals were sacrificed and the brains collected as described above. To isolate synaptosomes, the tissue was homogenized in Syn-PER synaptic protein extraction reagent (ThermoFisher). A portion of the total homogenate was saved for biochemical analysis and the remaining portion was centrifuged at 1200 × g for 10 minutes at 4 °C. The supernatant was collected and centrifuged further at 15000 × g for 20 minutes at 4 °C, as per reagent instructions. The synaptosomes containing pellet were resuspended in HEPES-buffered Krebs-like (HBK) buffer or radioimmunoprecipitation assay (RIPA) buffer depending on future experiment. The quality of the synaptosomal isolation is routinely verified by Western blot and electron microscopy, as shown by Franklin, 20162O. A cap with four holes was placed on the tube and the sample was stirred by a magnetic stir bar under a fume hood for 48 hours. The sample was used immediately after the 48 hours of stirring54. For the flow cytometry analysis of Aβ oligomer binding to the synaptosomes, Aβ oligomers with a Flour 488 tag were utilized. These Aβ oligomers were prepared by adding Aβ1–42 peptide with a Flour 488 tag  to the HFP-Aβ mixture described above. This mixture was then added to 0.7 ml of H2O and spun, as described. Western blot and dot blot analysis using A-11 antibodies (Aβ oligomer specific) are used to determine the quality of oligomerization (previously described by Reese (2008)55.Human Aβ oligomers were prepared from lyophilized synthetic Aβ aliquots (0.3 mg) dissolved in 0.2 ml of 1,1,1,3,3,3- Hexafluro-2-propanol (HFP). The HFP- Aβ mixture was then added to 0.7 ml of Hex vivo binding of Aβ oligomers to the synaptosomes of mice treated or not with NIR light was evaluated using flow cytometry. Synaptosomal fractions of male and female wild type mice receiving NIR light treatment were prepared and resuspended in HBK buffer. Synaptosomal fractions from multiple animals in each group were combined in separate tubes. Pooled samples were then aliquoted into 8 separate tubes per group containing 100 mg of total protein per tube determined by bicinchoninic acid (BCA) assay. To perform analysis using flow cytometry, the synaptosomes were prepared and aliquoted as described in the ELISA analysis, however, the synaptosomes were treated for 60 minutes with Aβ oligomers tagged with HyLite Fluor 488 (AnaSpec) in various concentrations ranging from 1 μM to 10 μM. The synaptosomes were then centrifuged and washed multiple times with HBK buffer and resuspended in PBS. Data was acquired by a Guava EasyCyte flow cytometer (EMD Millipore) and analyzed using Incyte software (EMD Millipore).The 1–42 specific solid phase sandwich enzyme-linked immunosorbent assay kit (ELISA) (Invitrogen).Synaptosomes and total homogenate were prepared from Tg2576 mice as described above and resuspended in RIPA buffer. Western blot analysis was performed on these protein extracts to determine the presence of Aβ oligomers. Separation of the proteins in the samples obtained was done by 12% SDS- polyacrylamide gel electrophoresis. The separated proteins were transferred to a nitrocellulose membrane (Bio-Rad) and incubated with 1:1000 6E10  antibody overnight. The nitrocellulose membrane was then incubated with the appropriate fluorescent secondary antibody and imaged with an Odyssey infrared imager. The band densities were analyzed using Image J software, normalizing using the densities of the loading control obtained by reprobing the membranes for β-tubulin. Quantification of Aβ levels in the protein extracts was determined using an Aβ2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 5 M sodium ascorbate, 2 mM thiourea, 3 mM sodium pyruvate, 10 mM MagSO4 ·7H20, 0.5 mM CaCl2 ·2H2O, and 12 mM N-acetyl L-Cysteine. Brains were harvested and sliced in the NMDG solution followed by a 10-minute recovery period in 35 °C NMDG solution. The slices were then maintained in a modified HEPES holding aCSF . For recording the slices where held in an artificial cerebrospinal fluid (aCSF)  . All solutions were supplemented with 95% O2/5% CO2. Prior to recording, the slices were incubated with 200 nM of Aβ oligomers for 1 hour at room temperature. Field excitatory post-synaptic potentials (fEPSPs) recordings were performed by stimulating the Schaffer collateral pathway. The stimulating electrode of 22 kΩ resistance was placed in the cornu ammonis 3 (CA3) region and the recording electrode was located at the junction of the alveus and cornu ammonius 1 (CA1). High frequency stimulation consisting of 3–100 Hz trains for 1 second duration with 20 second intertrain intervals was be used to induce LTP. The traces were analyzed with Clampfit 10.6 software (Molecular Devices).The wt mice were anesthetized with isoflurane and were perfused intracardially with a NMDG solution containing; 93 mM NMDG, 2.5 mM KCl, 1.2 mM NaH29. The fluorescent dye MitoSense correlates with mitochondria membrane potential30. The synaptosomes were washed twice with HBK buffer and fluorescent emittance was acquired by a Guava EasyCyte flow cytometer (EMD Millipore) and analyzed using Incyte software (EMD Millipore).The synaptosomes were isolated from frozen brain tissue as described earlier. The synaptosomes were then immediately treated with Mitotracker green FM (Invitrogen) and MitoSense Red  (EMD Millipore) for 15 minutes at 37 °C. MitoTracker is a fluorescent dye that diffuses across the mitochondrial membrane and reacts with thiol groups of specific mitochondrial proteinsThe statistical analysis of the data was analyzed using SPSS software. Student’s t test was used to determine statistical significance.Supplementary Information


The results suggest that the states having close energy levels and similar exciton characteristics with same transition configurations and high heteroatom participation are prone to facilitating exciton transformation. A basic guideline towards the molecular design of purely organic materials with facile exciton transformation ability is also proposed. Our discovery highlights systematically the critical importance of vertical transition configuration of excited states in promoting the singlet/triplet exciton transformation, making a key step forward in excited state tuning of purely organic optoelectronic materials.Exciton transformation, a non-radiative process in changing the spin multiplicity of an exciton usually between singlet and triplet forms, has received much attention recently due to its crucial effects in manipulating optoelectronic properties for various applications. However, current understanding of exciton transformation mechanism does not extend far beyond a thermal equilibrium of two states with different multiplicity and it is a significant challenge to probe what exactly control the transformation between the highly active excited states. Here, based on the recent developments of three types of purely organic molecules capable of efficient spin-flipping, we perform  This phenomenon of exciton transformation has been known for decades, but important details of its photophysical mechanism remain unrevealed, let alone the rational molecular design for controlled exciton transformation between the highly active excited states. Nevertheless, the interest in exciton transformation of purely organic molecules has been greatly renewed in recent years, because of the significant effects in promoting device performance by properly adjusting excited states for the desired forms of excitons. For example, highly luminescent singlet excitons are favorable for organic light emitting diodes (OLEDs)5, while long-lived triplet excitons are attractive for organic photovoltaics (OPVs) with long diffusion length to enhance exciton dissociation to generate free charges7 and for lifetime-resolved encryption and sensor applications with long-lived phosphorescence to eliminate the disturbance of short-lived background fluorescence9. Typically, by converting the electronically excited 75% triplet excitons to the singlet ones via RISC for thermally activated delayed fluorescence (TADF)10 or hybridized local charge-transfer (HLCT)11, a large amount of TADF  from purely organic materials has aroused much attention very recently with particular interests in luminescent mechanisms and molecule design principles of this novel kind of material16. Especially, organic ultralong RTP (OURTP) molecules  and triplet (T1) excited states offer an important path to understand the transformation mechanism. Both efficient ISC and RISC between S1 and T1 with rate constants up to ~106 and 104 s−1 respectively for exciton transformation have been experimentally identified in TADF materials and proved to be the key factor for their high OLED performance with external quantum efficiency (EQE) about 30%18. Another applicable guideline for enhancing ISC is El-Sayed rule19, which suggests that 1 → 3 ISC is faster than 1 → 3 transitions and 1 → 3 is faster than 1 → 3 transitions. This means the participation of heteroatom with either non-pair electrons or empty orbitals in the π-conjugated system is essential to promote the ISC process. Besides the energy gap law and El-Sayed rule, Golden Rule Approximation was also used to evaluate the exciton transformation. To facilitate the spin-flipping, the relativistic effect of spin orbital coupling (SOC) should be promoted through increased electromagnetic interaction between the electron’s spin and the magnetic field generated by the electron’s orbit around the nucleus20. The ISC rate (kISC) from an initial singlet state (1ψ0I) to a final triplet state (3ψ0F) can be described in the golden-rule expression , and L and S are the orbital and spin momenta, respectively. These calculations are rather complicated with considerable approximations, making the investigation of exciton transformation quite difficult, let alone giving a clear structure-property relation for rational control of both ISC and RISC in the molecular design of the related materials.The minimum requirement for realizing exciton transformation is the matching of the energy levels of the two states, based on a thermal equilibrium between the singlet and triplet excited states (energy gap law). Photophysical studies of TADF molecules with the small bandgap between the lowest singlet , similar exciton characteristics (containing the same transition configurations with high similarity) and high heteroatom participation are prone to exciton transformation for efficient ISC and RISC processes. This work can provide not only a facile theoretical method to evaluate the singlet/triplet exciton transformation, but also in-depth structure-property understandings into the exciton multiplicity manipulation.Here, we aim to investigate the crucial factors in determining the exciton transformation involved in three kinds of new-emerged purely organic optoelectronic materials of TADF, HLCT, and OURTP molecules capable of efficient exciton transformation. Density functional theory (DFT), time-dependent DFT (TD-DFT), and natural transition orbital (NTO) calculationsDMAC-DPS26 and Spiro-CN27), two HLCT (MADa29 and TPA-NZP29), and two OURTP (DPhCzT and DCzPhP17) molecules, which have been experimentally found to have efficient exciton transformation properties, were selected to investigate the exciton transformation principles at single molecular states  and triplet (ETn) excitation energies, and singlet–triplet energy splitting (ΔEST) at excited states, we tested various DFT methods including B3LYP, PBE0, BMK, M06-2X, M06-HF that have different Hartree-Fock hybrid proportions, and long-range correction functional of ωB97XD at 6-31 G(d) basis set30. By comparing to the experimental results, the most applicable functionals for TADF, HLCT, and OURTP molecules are PBE0, M062X, and PBE0, respectively  moieties with large twisting angles (88.3° and 89.9° respectively), which is important to support the strong charge transfer (CT) feature with small HOMO-LUMO overlap extent (IH/L) and single-triplet splitting between S1 and T1 (ΔEST11) for efficient ISC and RISC processes31. In the HLCT molecules of MADa and TPA-NZP, the twisting angles are decreased to 42.4° and 56.1° with large IH/L (61.7% and 46.6%) to harmonize the hybridization of locally excited (LE) and CT states for efficient fluorescence with large transition moment and weakly bound exciton with facile exciton transformation at high-lying excited states, respectively32. Whereas, the twisting angles between the D (carbazole) and A (triazine and phenyl phosphine) moiety planes of OURTP molecules can be either large (68.5° in DCzPhP) or small (20.1° in DPhCzT) with varied IH/L of 47.4% and 24.3%. Therefore, although the separated HOMO and LUMO distribution on donor and acceptor moieties with small IH/L is important for TADF molecules to achieve small ΔEST11 with facilitated ISC and RISC processes, efficient singlet/triplet exciton transformation can also occur as found experimentally in HLCT and OURTP molecules with large ΔEST11 and IH/L up to 1.26 eV and 61.7%, respectively.At the ground states Figs  and S1, 33; below this energy gap, the transformation can be compensated or dissipated through vibrations of the molecules at room temperature in a thermal equilibrium. For TADF molecules, exciton transformation happens between S1 and T134, while for HLCT and OURTP molecules, the transformation is from the high-lying Tn to Sm (n > 1 and m ≥ 1) and from S1 to Tn (n > 1), respectively35. Principally, all of these transformations require a small ΔEST. However, to support the high-lying excited state-related transformation as in HLCT and OURTP molecules, the separated HOMO-LUMO distribution strategy in achieving small ΔEST fails. Therefore, it is difficult for frontier molecular orbital analysis to give an overall and convincible description of the exciton transformation processes in HLCT and OURTP molecules.According to the energy gap law, the initial and final states for the exciton transformation should have similar energy levels and the energy difference must be lower than 0.37 eVvia TD-DFT calculations  → LUMO (L) components; there are also apparent component overlap with the same transition configurations between S0 → Sn (n = 1 and 2) and S0 → T1 in HLCT molecules of MADa and TPA-NZP; in OURTP molecules (DPhCzT and DCzPhP), still the same transition configurations can be observed between S0 → S1 and S0 → Tn  within the critical energy gap of 0.37 eV. Considering the El-Sayed rule that predicts accelerated ISC by vibronic interactions between  and  states19, the same transition configuration component of the two excited states indicates their overlapped excitation features, which should be crucial for the enhanced ISC through these allowed transformation channels; and it can be expected that the more overlapped the transition configurations are, the more facile the exciton transformation will be through this channel37.We then performed transition configuration investigation of the excited states 1 and T1 are very similar in TADF molecules; for instance, DMAC-DPS has the same transition configurations of 6.4% and 6.8% in H-1 → L + 2 and of 91.1% and 90.4% in H → L for S1 and T1 respectively, suggesting the very efficient ISC and RISC processes at room temperature as found experimentally26. For HLCT molecules, the configuration overlap between T2 and Sn (n = 1 or 2) is also high, which is over 90% (64.5% + 27.6%) from the sum of the two lower transition configurations in MADa and 77.0% (34.4% + 42.6%) in TPA-NZP for T2 → S2; it should be noted that a high energy barrier between T2 and T1 states is required to hinder the rapid internal relaxation to maintain the stable T2 with high exciton population for RISC11. In OURTP molecules, overlapped transition configuration between S1 and Tn was obvious too, reaching 93.9% in DPhCzT and 29.5% in DCzPhP respectively; the lower configuration overlap in DCzPhP may be a reason for its relatively weaker organic afterglow emission17.Specifically, the transition configurations of SEST lower than 0.37 eV, otherwise the transformation cannot be fueled by the environmental thermal energy at room temperature or facilely disperse the excess energy via molecular vibrations. Thirdly, the two excited states should contain the same components of transition configurations to establish the transformation channels in bridging the spin-forbidden transitions between two electronic states with different spin multiplicities. Without the matched energy gap, the excitons are weak in transformation due to the lack of enough energies achievable from thermal vibrations or suitable ways in dissipating excess energies. Without the matched transition configuration, the excitons are locked by the forbidden spin state change for transformation. Only when both the matched energy gap and transition configuration with high heteroatom participation are satisfied, facile exciton transformation via ISC and RISC processes is possible in purely organic optoelectronic molecules.Taken both the energy gap and transition configuration together, a universal exciton transformation mechanism for TADF, HLCT, and OURTP molecules can be proposed Fig. . Firstly0)25. HONTOs and LUNTOs of all the studied six molecules at the lowest 10~15 singlet and triplet excited states were investigated   or overlapped (DCzPhP), but they should be similar enough at the two singlet and triplet excited states, i.e. the HONTO (and LUNTO) at S1 is well overlapped with HONTO (and LUNTO) at T1, to support the exciton transformation channels for ISC  analysis based on the singular value decomposition of 1-particle transition density matrix was performed. The resulting compact frontier orbitals can represent any one electron property associated with the electronic transition. In principle, the highest occupied natural transition orbital (HONTO) to the lowest unoccupied natural transition orbital (LUNTO) excitation amplitude is always the most significant for any particular excited state, due to its dominating role in determining the one electronic transition for the generation of the corresponding excited state from the ground state  Fig. , very sites Fig. . As to OISC Fig. .Figure 5n (IS) and Tn (IT) states were calculated using Multiwfn39. The separated HONTO and LUNTO distributions lead to small IS and IT, while large IS and IT represent heavily overlapped HONTO and LUNTO. Interestingly, small IS and IT were observed in TADF molecules; larger IS and IT were found in HLCT molecules; but OURTP molecules show both small and large IS and IT. The key point is that IS and IT should be close in value for all the three kinds of materials, suggesting that the close values of IS and IT are a direct result of the similar HONTO and LUNTO distributions at singlet and triplet excited states. Based on these values, CT (0~40%), HLCT (40~75%), and LE (75~100%) characters of both singlet and triplet excited states can be quantitatively classified. In this sense, both S1 and T1 need to be in CT with low IS and IT for TADF molecules; HLCT character of Tn (n > 1) with moderate IT is essential for HLCT molecules to transform to singlet excited states with both CT and LE characters, since HLCT contains both LE and CT components29. For CT characterized OURTP molecule of DPhCzT, both S1 and T4 should be CT with low IS and IT, while for HLCT type OURTP molecule of DCzPhP, the HLCT characterized S1 offers many transformation channels to all the CT, HLCT, and LE characterized triplet excited states with close IT values to IS.Furthermore, in order to give a quantitative evaluation of HONTO-LUNTO overlap, the overlap extents at both Ss):iai = 1 and Σibi = 1. The index i is the number of atoms in the molecule; ai and bi are the contribution percentages of different atoms in the frontier NTO of the corresponding singlet and triplet excited states, respectively40. This orbital composition analysis was done using Multiwfn. |ai − bi| denotes the contribution percentage difference of an atom (i) in HONTO (or LUNTO) between the singlet and triplet excited states.Since the HONTO and LUNTO describe the transition features of an excited state in a whole, similar HONTO and LUNTO between the singlet and triplet excited states means the similar excitation feature, which would essentially provide effective channels for the exciton transformation between them. Therefore, we developed a new formula in equation  to evalusH and sL, which describe quantitatively the orbital similarity in HONTO (sH) and LUNTO (sL), offer new parameters in evaluating the exciton transformation of organic molecules. Large values of sH and sL indicate high similarity of HONTO and LUNTO between the singlet and triplet excited states. For TADF, HLCT, and OURTP molecules capable of efficient exciton transformation as found experimentally, both high sH and sL should be observed between singlet and triplet excited state pairs with energy gap below 0.37 eV33; and the larger values of sH and sL, the more facile the transformation channels will be. With this basic consideration, possible exciton transformation channels in all the studied molecules can be picked out by scanning both the energy gap and transition similarity (sH and sL). Since higher s means more facile intersystem crossing channel, it should be reasonable that the values of sH and sL must be both larger than 0.50 for efficient exciton transformation. To our delight, the predicted possible exciton transformation channels are well in line with that obtained through above transition configuration analysis via TD-DFT calculations, except for S1 → T8 of DCzPhP and T2 → S2 of MADa  to support the efficient exciton transformation. These two differences are understandable, because the S1 → T8 transformation channel in DCzPhP must be very weak with H → L ratio of only 3.1%, while in the case of T2 → S2 of MADa, its energy gap is high up to 0.34 eV, suggesting the high energy barrier in exciton transformation via this channel.The ADa Fig. . The tra1 and T1 in TADF molecules, but in HLCT and OURTP molecules, the transformation channels cannot be easily identified, although the ISC and RISC do proceed efficiently from experimental evidences. With our new-proposed parameters, it is possible to describe the excited state similarity quantitatively and more interestingly, to figure out the transformation channels and their feasibility. From Fig. sH and sL > 0.50) to support both ISC and RISC processes in these molecules. Four channels  with very high sH and sL (>0.95) are found to be available for the facile exciton transformation in the TADF molecule of DMAC-DPS; this should be an important reason for its high TADF performance in device applications26. The highest sH and sL found in TADF molecules suggest the existence of the facile transition-allowed channels for both ISC and RISC, which is in line with the experimental results (EQE≈30%)10. In HLCT molecules, sH and sL are slightly reduced, and they can be either high or low in OURTP molecules, suggesting relatively lower exciton transformation efficiency in these molecular systems; this understanding well explains the lower OLED performance (EQE≈8%) of HLCT molecules41 and the weak ultralong-lived phosphorescence of OURTP molecules observed experimentally42.It is clear that both ISC and RISC happen between SDPhCzT of various transformation channels from S1 to Tn were calculated using Dalton package (B3LYP/cc-pVTZ) based on its optimized S1 state structure. From Supplementary Table 1 → T3 and S1 → T4 channels have relatively high SOC values, which is matched with our predictions of S1 → T4. Although our qualitative method cannot predict the high SOC of S1 → T3 suggested by the Dalton calculation, our method succeeds in figuring out efficient SOC channels for exciton transformation. To further identify the proportion of  configuration (αn%) of the excited states, Mulliken population analysis (MPA) was performed on the optimized excited state structures with the aid of Multiwfn package16. The S1 of DPhCzT was found to have a high component of 1 with αn% of 14.3%, while the T4 is mainly 3 with αn% of 0.0%; Such a significant αn% change  suggests the allowed 1 → 3 transition from S1 to T4 according to El-Sayed rule, which is also coincident with the results of our TD-DFT calculation method based on the ground state geometry (S0).To test the validity of our method in predicting the exciton transformation channels and efficiencies, the SOC values of 43, Compound 1 has very slow intersystem crossing rate (kISC), while the exciton transformation in Compound 2 is very efficient (Supplementary Table 1 → T3 transformation channel (2.92%) can be observed in compound 1, while Compound 2 has three available channels and that of S1 → T1 and S1 → T5 transformations are highly efficient with high transition configuration component overlap   were optimized via spin-restricted formalism at the B3LYP/6-31 G(d) level of theory. Vibrational frequency calculations were carried out to test that the optimized structures are truely corresponding to the minima on the potential energy surfaces. The excited singlet (Sn) and triplet (Tn) states were investigated by the time-dependent DFT (TD-DFT) formalisms of B3LYP (20% HF), PBE0 (25% HF), BMK (42% HF), M06–2X (56% HF), and M06-HF (100% HF) functionals with 6–31 G(d) basis set were performed based on the optimized ground-state geometries to investigate the vertical excited energies. The vertical excitation energies of the studied molecules were also evaluated using the range separated hybrid exchange functional of ωB97XD, which consists in a mix of short range density functional exchange with long range Hartree-Fock exchange (22% HF at short range and 100% at long range). To obtain a precise picture of the excited states, natural transition orbitals (NTOs) analysis were further performed to give a compact orbital representation for the electronic transition density matrix. The SOC values between S1 and Tn were calculated using Dalton package (B3LYP/cc-pVTZ) based on the optimized S1 state structure. The proportion of 1 configuration (αn%) of the excited state was calculated by using Mulliken population analysis (MPA) to identify the n orbital components of the frontier orbitals at the optimized excited state geometries with the aid of Multiwfn package16.DFT and TD-DFT calculations were performed to investigate the singlet/triplet exciton transformation using Gaussian 09 package. The molecular geometries in the ground state (SSupplementary information


Rora allele that we crossed with a mouse line expressing Cre recombinase under the control of the EIIa promoter to obtain a full body deletion of Rora. This cross led to a partial deletion of the Rora locus likely due to mosaic expression of the EIIa-Cre transgene. These mice lack any signs of ataxia but display an improved metabolic profile relative to normal WT mice. The mice were resistant to diet- and age-induced metabolic syndrome and exhibited improved glucose tolerance and increased insulin sensitivity. Decreased RORα expression in the mice was also associated with reduced inflammation in models of metabolic syndrome. These data indicate that suppression of RORα activity improves metabolic function and reduces inflammation.The retinoic acid receptor-related receptor α (RORα) is a nuclear receptor that plays an important role in regulation of metabolism and the immune system. Genetic deletion of the receptor yields mice with significant cerebellar developmental issues associated with severe ataxia. Although many metabolic studies have been performed in these models, the impaired locomotor activity of these mice is known to affect their normal mobility and feeding behaviors. This creates some difficulty in interpretation of the role of RORα in models of metabolic disease where feeding and muscle function is a critical component of the pathophysiology. We generated a mouse with a floxed  More than two-thirds of American adults are considered to be overweight or obese. Obesity is associated with a range of diseases including diabetes, cardiovascular diseases and several cancers. Obesity is the result of a positive energy balance, a consequence of excess caloric intake relative to calories expended. Energy expenditure is the combination of resting metabolic rate (energy consumed at rest), the thermogenic effect of food (energy utilized to metabolize food consumed), and energy expended during physical activity. When energy expenditure exceeds energy intake, it results in a negative energy balance leading to weight loss. The mechanisms underlying the regulation of energy balance are not completely understood, but evidence suggests that complex physiological regulation and genetic predisposition are involved . In addiin vivo using the staggerer (RORαsg/sg) mouse model (Nuclear receptors (NRs) are ligand-dependent transcription factors that regulate many biological functions including development, metabolism, and inflammation . In the se model , 8. In sse model .Rora allele that we crossed with a mouse line expressing Cre recombinase under the control of the EIIa promoter to obtain a full body deletion of Rora. As the EIIa-cre model induced a mosaic expression of the cre recombinase, we did not obtain a total recombination of the Rora Flox loci but a partial deletion. These animals (EIIaRORα KO) lack any signs of ataxia but display an improved metabolic profile relative to normal EIIaRORα WT mice. Our study presents a new model to study the role of RORα in metabolic disorders without the complexity of a cerebellar deficit that may alter normal feeding behavior and physical activity. As RORα can be targeted by synthetic ligands . Tissues were collected, snap frozen and stored at −80°C for qPCR or western blotting, or fixed in 4% formalin, incubated in 20% sucrose overnight before freezing for cryosection.All mice were housed in a 12-h light/dark cycle. At the end of the experiment, mice were euthanized by COrora gene. Female Roraflox/flox mice were bred with male mice homozygous for the Cre recombinase gene under the control of the adenovirus EIIA promoter to generate the Rora+/ΔCre F1 generation. EIIa-Cre transgenic mice were obtained from The Jackson Laboratory . Genotypes were determined by PCR using the oligonucleotide primers: primer 1: 5′-GCCCCCTTTCCCGCCAGTAGCTG-3′; primer 2: 5′-GACCCAGATCCCCTAATAACG-3′; primer 3: 5′-GCCCTCAAGAAGAGGCTGCAATTT-3′  by inserting LoxP sites by homologous recombination flanking the exon three of the 2, VCO2, heat production, and activity were measured using a monitoring system . Eight aged-matched male mice (EIIaRORα WT and EIIaRORα KO) were housed individually in metabolic cages on a 12 h day–night cycle, fed with either a normal chow or high fat diet (HFD) . After a 24-h acclimatization period, data were collected every 16 min over a 5-day period. The hourly or average values during light and dark periods were calculated. Two-way ANOVA followed by Bonferroni posttest was used to calculate the p value.Respiratory exchange ratio (RER), VOn = 8) were injected intraperitoneally with glucose (2 g/kg of fat free mass) or insulin (0.75 U/kg of fat free mass)  to examine glucose tolerance test or ITT, respectively. Blood glucose was measured before the injection (t = 0 min), 15, 30, 60, and 120 min after injection using OneTouch Ultra®2 glucometer. Blood was collected by tail snip; the first drop was wiped out, and the second drop was used to quantify glucose level.After 6-h fast, EIIaRORα WT and EIIaRORα KO  cholesterol, LDL cholesterol, triglyceride, and liver enzymes were assessed using Rx Daytona clinical chemistry analyzer (Randox). Plasma TNFα or IL-1β concentrations were detected by ELISA  according to the manufacturer’s recommendations.Mice were euthanized, and blood was collected Total RNA was isolated from mouse tissues using PureLink RNA mini kit . RNA was reverse transcribed to make cDNA using qScript™ cDNA Synthesis Kit (Quanta biosciences) according to the manufacturer’s instructions. Real-time PCR was performed using a SYBR-green PCR master mix kit . Primers were purchased from Integrated DNA Technologies .g) at 4°C. Cell pellet was resuspended in Flow Cytometry Staining Buffer  at the final concentration of 2 × 107 cells/mL. Cell surface and intracellular staining were performed according BioLegend protocol (BioLegend). Antibodies used for analysis are listed in Table S2 in Supplementary Material.Spleens and lymph nodes were harvested into a tissue culture dish and teased apart into a single cell suspension. Cell suspension was passed through a cell 0.22 µM filter (EMD Millipore) and centrifuged ]. Roraflox/flox littermates without the EIIa-Cre transgene (EIIaRORα WT) served as controls. When we assessed the level of Rora expression in different tissues, we found that the deletion was not complete. Rora expression ranged from 45 to 30% of normal levels depending on the tissue examined. We observed a significant decrease of expression in several tissues: 65% in white adipose tissue (WAT), 65% in liver, 70% in brain, 55% in BAT, and 60% in the skeletal muscle  were fed with a chow diet and used to decipher the role of RORα in energy and glucose metabolism. Whole body composition was determined weekly using NMR , and no significant differences were observed in body weight, lean mass, or fat mass Figure A. Using Three-month-old male mice were challenged with an HFD  over 14 weeks and monitored for body composition weekly, fuel utilization (at week 1 of HFD) and glucose metabolism. EIIaRORα KO mice exhibited resistance to DIO displaying significantly reduced body weight gain and reduced fat mass relative to control mice Figure A. After reg), was increased in EIIARORα KO animals compared to EIIARORα WT suggesting more infiltration of Treg cells  was observed in old-EIIARORα KO mice compared to old-EIIARORα WT mice as well as a decrease of pro-inflammatory lymphocytes TH17  that will promote Treg differentiation and block TH17 differentiation.In this study, we have described a novel mouse model that we used to characterize the role of RORα in metabolic disorders. These EIIARORα KO mice, which expressed significantly reduced RORα expression relative to WT mice but did not display mobility/ataxia issues typically associated with loss of RORα function, were protected against diet- and age-induced metabolic syndrome. In these mice, reduction of RORα expression protected against DIO and improved glucose metabolism and insulin resistance were observed while also decreasing markers of inflammation. Our study extends previous ones utilizing the staggerer mouse model that also suggest a metabolic benefic of RORα deletion , 8. Howentiation , 5. Our how diet . HIF-1α  by RORα . We hypoAged-EIIARORα KO or EIIARORα KO mice under HF diet for weeks are still insulin sensitive and glucose tolerant, suggesting that these mice are less susceptible to aged or diet-induced type-2 diabetes. Very interestingly, we observed an upregulation of Akt signaling in the muscle of EIIARORα KO animals, which could explain the more efficient insulin signaling. Further studies regarding the insulin pathway will be necessary to understand the role of RORα deletion in this process. The mechanisms underlying these results are not clear, but it could be a link to a decrease of inflammation and a better peripheral glucose metabolism. The muscles play a critical role in glucose metabolism and insulin sensitivity . PreviouObesity and fat accumulation are the results of imbalance between energy intake and expenditure. Indirect calorimetry analysis of 2-month-old EIIARORα KO and EIIARORα WT fed with normal chow diet as well as aged EIIARORα KO and EIIARORα WT have shown an increase in energy expenditure and fatty acid oxidation without alteration of food intake during both night- and daytime. We hypothesized that the increase in energy expenditure was linked to an increase in thermogenesis in the brown adipose tissue (BAT). To test this, we challenged young EIIARORα KO and EIIARORα WT to cold temperature, but no differences were observed in energy expenditure, fatty acid oxidation, or BAT activity (data not shown). We then challenged EIIARORα KO and EIIARORα WT mice with HF diet for 8 weeks before performing the same indirect calorimetry analysis. As EIIARORα KO mice accumulate less fat, the normalization to the fat free mass excludes any weight effect in energy expenditure. EIIARORα KO mice are utilizing more energy during both day- and nighttime and have an increase in fatty acid oxidation. Fatty acid oxidation has been shown to be impaired in aging animals , but ourIn conclusion, we developed a new RORα deficiency mouse model that recapitulates the observations performed in the Staggerer mice but without the problematic effects of ataxia. Partial deletion of RORα protects against diet- and age-induced metabolic syndromes and inflammation. This RORα floxed model will provide a new tool to study the role of RORα in metabolic syndrome. By generating tissue-specific RORα deletions using this model, we will be able to better understand the role of RORα in metabolic tissue and some of the problematic cerebellar developmental effects. We previously published that ROR can be targeted by synthetic ligands and protects against inflammation, diabetes, and cardiovascular diseases , 5, 18, All animal studies were approved by the SLU IACUC.TB and CB conceptualized the project; wrote and edited the paper. CB and SS performed experiments. All the authors analyzed the data.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


N‐heterocyclic carbenes (NHCs) on copper are reported. The monolayer structure is highly dependent on the N,N‐substituents on the NHC. On both Cu(111) and Au(111), bulky isopropyl substituents force the NHC to bind perpendicular to the metal surface while methyl‐ or ethyl‐substituted NHCs lie flat. Temperature‐programmed desorption studies show that the NHC binds to Cu(111) with a desorption energy of Edes=152±10 kJ mol−1. NHCs that bind upright desorb cleanly, while flat‐lying NHCs decompose leaving adsorbed organic residues. Scanning tunneling microscopy of methylated NHCs reveals arrays of covalently linked dimers which transform into adsorbed (NHC)2Cu species by extraction of a copper atom from the surface after annealing.Self‐assembled monolayers of  In thiolate SAMs on gold, solution deposition methods give N‐heterocyclic carbenes (NHCs) have emerged as promising alternatives to thiols for the formation of robust, ordered SAMs on Au.Recently, Despite the impact of these studies, the effect of NHC structure on SAM formation has barely been examined. Additionally, the ability of NHCs to form monolayers on other, more reactive metals has received no attention outside the realm of nanoparticles.Herein we present the preparation of highly ordered, thermally stable NHC films on Cu(111). Through STM, high resolution electron energy loss spectroscopy (HREELS), and temperature programmed desorption (TPD) studies, we demonstrate that small differences in the size of the NHC wingtip groups lead to two distinct binding modes. In addition, we find that NHCs bind to Cu(111) with a similarly high adsorption energy to that observed on Au(111).1 to 3) were vapor deposited onto clean Cu(111) and Au(111) surfaces  and Au(111) were dominated by a very strong peak at 730 cm−1, assigned to the out‐of‐plane aromatic C−H bending mode, whose dipole moment is normal to the molecular plane. Features at 2940 and 3075 cm−1  and at 1250–1600 cm−1  all appear very weak. The relative intensities of these energy losses provide strong evidence that the molecular planes of 1 and 2 are aligned approximately parallel to both the Au(111) and Cu(111) surface planes.  led to significant enhancement of the weaker signals, implying an increasing amount of upright species at higher coverage . However, the 730 cm−1 peak was not suppressed; therefore, the spectrum reflects the coexistence of upright and flat‐lying species. Such behavior is not uncommonProlonged exposure of NHC 3 on Cu(111) and Au(111) are significantly different from those obtained for NHCs 1 and 2, with strong peaks observed with 1 and 2 appearing weak for 3, and vice versa .Spectra recorded for the bulkier NHC 3 on Cu11 and Au1 on Cu(111) at 300 K, along with trenches of a depth equivalent to a single Cu atom. Elliptical features of length 3.4±0.7 Å, were resolved in the trenches =77.6°. These dimensions conform with a commensurate (7 4|1 10) superstructure =80.08°, 2AuI complexes.2Au complexes containing less sterically bulky NHCs favor flat lying NHC geometries driven by vdW interactions between the NHCs and the Au surface.2Cu0 complexes have been isolated and characterized.1 on Au(111) , though the species were too mobile to image clearly. No ordering of NHC 3 was found on either Cu(111) or Au(111) at any coverage when imaging at 300 K. It is likely that the upright NHCs exhibit similar “ballbot‐type motion” to that described by Glorius and Fuchs for NHCs on Au.Extraction of Cu atoms from steps and incorporation into molecular assemblies is a thermally activated process.upright NHC 3 desorbed cleanly from both Cu(111) and Au(111), flat‐lying NHCs 1 and 2 underwent more complex decomposition and desorption processes . Figure m/z=39 (C3H3+ from the benzene moiety) following the adsorption of NHC 3 onto Cu(111) at 300 K.TPD data further confirmed that the two types of adsorbed NHCs display distinctly different behavior. While max=570 K) independent of coverage; typical of first‐order kinetics.2, HCN, and C3H5+ (assigned to isopropyl groups) were also detected , suggesting that NHC 3 desorbs intact from Cu(111). A Redhead analysis yields a desorption energy of 152±10 kJmol−1; indistinguishable within error from the reported value of 158±10 kJmol−1 on Au(111).3 species on a Cu(111) slab predicts a binding energy higher than that observed on Au (182.6 kJmol−1 (see SI)). However, errors in such calculations are typically ≈20 kJmol−1, so theory and experiment are mutually consistent within error. Repulsive lateral interactions  may explain the lower value measured by TPD, though the fact that Tmax is independent of coverage points to such interactions being relatively weak. These results confirm that NHCs form highly stable SAMs on Cu surfaces.Desorption occurred in a single peak  occurs in two overlapping peaks at 532 K and 590 K which can be assigned to sequential thermally activated dehydrogenation steps.m/z=27) occurs concurrently with H2 evolution, signifying that these desorption events are related. Unlike the TPD spectra for NHC 3, the relative intensity of the fragments did not correlate with the exposure, suggesting a more complex surface chemistry. The yield for m/z=39 is marginal, implying that the ring moiety remains adsorbed  up to the onset temperature for the first dehydrogenation step (≈475 K), contrasting starkly with the behavior of NHC 3.For films derived from NHC d Figure  b. It ca3 films resulted in clean surfaces, consistent with NHC desorption via a simple C‐Cu bond cleavage  resembled closely the behavior observed for 1, and an analogous interpretation is proposed. Thermal stability of the SAMs was also assessed by HREELS . Overall attenuation of all spectral features occurs above 560 K for all NHCs, consistent with the TPD findings.STM imaging of Cu(111) terraces after heating NHC e Figure  c. In cos Figure  d. TPD aN,N‐positions of the NHC are critical in determining the adsorption geometry and fate of the NHC‐based SAMs on Au and Cu. Small NHCs with dimethyl substituents form films with mixtures of flat and upright orientations, while the diisopropyl NHC stands upright only. The adsorption energy of NHC 3 on Cu(111) was found to be the same as on Au(111) within error. Upright NHCs desorb cleanly, while flat‐lying NHC films dissociate leaving surface contamination. For NHC 1, ordered arrangements of (NHC)2Cu complexes were imaged. The fact that appropriately designed NHCs bind to Cu with high bond energies is a significant discovery and paves the way for future work on the practical applications of this surface functionalization. This work is currently ongoing in our laboratories.In conclusion, the substituents at the See the Supporting Information for experimental details.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.