Top Panel Research Articles

Characterization Of Liver Metabolism By NMR‐Guided GC‐MS Methodology

Non‐Alcoholic Fatty liver disease (NAFLD), Non‐alcoholic Steatohepatitis (NASH), liver cirrhosis and liver cancer affect a combined 80–100 million Americans [Perumpail et al. World J Gastroenterology. 2017, 23(47), 8263]. In order to better diagnose these conditions and monitor treatment progression, several metabolomics approaches have been developed. Amongst these, Mass Spectrometry (MS) and Nuclear Magnetic Resonance (NMR) have been used extensively.In this study, a recently developed method of metabolic profiling coined NMR‐Guided LC‐MS is assessed for its ability to accurately measure metabolites. [Nagana Gowda et al. Anal. Chem. 2018, 90(3), 2001]. The principle of NMR‐Guided MS is to obtain a ratio of a metabolites mass spectral intensity to its assigned concentration from 1H NMR spectra in a reference sample. This ratio is then applied to mass spectral intensities for in other samples to assign a metabolites concentration. For NMR‐Guided MS to be applicable to a metabolite of interest the variance between NMR‐Guided MS and NMR concentrations must be low and constant over a concentration range of biological interest. In this case, we apply a similar normalization methodology to GC‐MS to assess the technical possibility of producing quantitative GC‐MS approaches to metabolomic analysis.To achieve the biological range of interest we extracted metabolites from ex vivo C57/BLKS perfused mouse livers under fed and fasted conditions. Additionally, two common extractions methods, perchloric acid extraction (PCA) and acetonitrile‐Isopropanol‐water (ACN/ISO/H2O) were compared.A significant fraction of high concentration compounds, including lactate and alanine, amino acids such as aspartate and glycine, and some TCA cycle intermediates such as succinate had low variance across fed and fasted samples between NMR‐Guided GC‐MS and NMR. Branched chain amino acids had greater variance by NMR‐Guided GC‐MS than GC‐MS alone when compared to NMR. In the case of β‐Hydroxybutyrate (BHB), the variance between NMRGuided GC‐MS and NMR was acceptable when extracted by PCA but not ACN/ISO/H2O for both tissue extracts and standard mixtures. BHB concentrations assigned by GC‐MS have a high variance relative to NMR regardless of the extraction method. However, for the majority of metabolites, differences in variance between extractions and assignment methods were subtle for livers and apparent only at concentration extremes of standards (Figure I).Altogether the data presented here provides insight about the comparability of GC‐MS and NMR systems, with excellent agreement observed between the methods across most metabolites. We have also demonstrated measurable extraction artifacts between PCA and ACN/ISO/H2O methods for some metabolites. In conclusion, NMR‐Guided GC‐MS may lend to more rapid and accurate metabolite measurements, broadening the swatch of applicability of the relatively inexpensive GC‐MS platform to metabolomics profiling.Support or Funding InformationNIH R01DK105346% coefficient of variance (% CV) for assigned concentrations by GC‐MS or NMR‐Guided MS compared to NMR assigned concentrations are presented as heat maps. The top panel represent standard samples across a range of 1 to 18 μg per metabolite extracted by PCA or ACN/ISO/H2O. While the bottom panel represents fed and fasted liver tissues extracted by PCA or ACN/ISO/H2O. Histograms in the middle provide density information on the %CVs and are color matched to their respective panels.Figure 1

Computational Model to Predict Tissue Concentration of Drugs Delivered via Intravital Fluid Exchange Devices

The microcirculation is capable of precise blood flow regulation enabling fine control over the distribution of oxygen (O2) to tissues in the body. The objective is to determine the role of capillaries in sensing and reporting local O2 concentration within tissue to upstream arterioles. It is hypothesized that red blood cells (RBCs) release ATP into the vessel lumen following O2 desaturation; ATP in the plasma binds to purinergic receptors on the endothelium triggering a signalling cascade that leads to a conducted vasodilatory response. Luminal application of ATP is known to initiate conducted dilation in arterioles yet whether or not this conducted response is present in capillaries has yet to be demonstrated.To investigate this mechanism of vascular control, we developed gas and liquid microfluidic devices for use in intravital video microscopy experiments to enable the local control of gases and agonists in skeletal muscle. These devices consist of a microfluidic flow channel interfaced with a glass slide with a laser cut micro‐outlet to allow exchange between the device and skeletal muscle during in vivo experiments. Experimentally, the gas exchange device was used to control the spatial distribution of O2 in the tissue to cause a highly localized hypoxic condition, to induce the release of ATP from RBCs. The liquid device was used to introduce ATP to a localized region of the capillary bed to stimulate ATP mediated mechanisms downstream. Further, we used acetylcholine (ACh) and phenylephrine (PE) as positive and negative controls, respectively. All animal protocols were approved by Memorial University’s Institutional Animal Care Committee.To quantify the local distribution of solutes in skeletal muscle we developed a transient, 3D transport model to predict the concentration of solutes applied experimentally in skeletal muscle. The transport equation was discretized and solved in parallel on a graphics processing unit. The computational model predicts highly localized spatial distributions of these solutes. As expected, the highest concentration of simulated solutes was at the surface of the tissue in contact with the exchange window, with steep decreases in concentration with increasing distance from the exchange window, falling by two orders of magnitude within 240 μm of the edge of the window in both the x and y directions, and by 300 μm in the z direction (see Fig. 1& 2). Logarithmic increases in applied drugs within the simulated microfluidic channel raise relative tissue concentrations proportionally. Predicted steady state concentrations of ACh, PE, and ATP varied due to the assumed rate of enzymatic degradation and efflux into the vasculature.In conclusion, our novel intravital video microscopy devices allow the exchange of gas and drugs to tissue to investigate the mechanisms of regulation in the microcirculation. Further, we quantified the localization of our devices using computational models of molecular transport, confirming the highly‐localized concentration gradients.Support or Funding InformationProject funded through NSERC Discovery Grant to GM Fraser.Tissue model results for 15 mmHg oxygen. The colour map shows the predicted tissue concentrations with 15 mmHg oxygen at the micro‐outlet. The top panel shows the gradients at the surface of the tissue closest to the gas‐based microfluidic device, in the x–y plane. The bottom panel illustrates the tissue partial pressure of oxygen perpendicular to the x–z plane centred at the middle of the micro‐outlet.Figure 1Tissue model results for adenosine triphosphate 10−4 M. The log scale colour map shows predicted tissue concentrations with 10−4 M adenosine triphosphate (ATP) at the micro‐outlet. The top panel shows the gradients at the surface of the tissue at the micro‐outlet. The bottom panel illustrates tissue ATP levels in the x–z plane centred on the micro‐outlet. ATP levels within the volume were above 3.47 × 10−7 M due to the initial assumption of blood ATP concentration.Figure 2

Effect of a lateral square-mesh panel on the catch pattern and catch efficiency in a Mediterranean bottom trawl fishery

The current legal codends used in Mediterranean bottom trawl fisheries are at high risk of retaining undersized individuals of several commercial fish species. This entails that codends alone are unable to provide the desired exploitation pattern. A simple technological measure that potentially can provide higher release efficiency of undersized fish are Square-Mesh Panels (SMPs). SMPs are often applied in the upper section of the trawl belly, just ahead the codend. However, recent studies in the Mediterranean have demonstrated that SMPs mounted in this position provided limited release efficiency, because very few fish came into contact with their meshes. In attempt to improve SMPs efficiency in the Mediterranean bottom trawl fisheries, we applied them on the lateral sides of the last tapered section of the trawl belly, just ahead of the codend, and fitted two guiding panels in the trawl belly to enhance fish-SMP contact. We compared the catch performance of a standard commercial trawl with a 52 mm diamond-mesh codend and of a similar trawl fitted with lateral SMPs (70 mm mesh size) in the belly using a twin trawl. The study focused on red mullet (Mullus barbatus), a commercially important species, but data for gurnard (Chelidonichthys lucerna and Chelidonichthys cuculus) were also obtained and analysed. In contrast to previous research on SMPs mounted in the top panel of the trawl, in this study SMPs induced a significant effect on catch performance for red mullet, demonstrating that their lateral position involved greater fish-SMP contact. However, since the test trawl lost a significant amount of legal-sized red mullet compared with the standard trawl, the effect was not wholly positive, possibly due to an excessively large mesh size. Therefore, future studies should be encouraged to test lateral SMPs with smaller mesh sizes.

Histopathologic Changes and SARS-CoV-2 Immunostaining in the Lung of a Patient With COVID-19

Histopathologic Changes and SARS-CoV-2 Immunostaining in the Lung of a Patient With COVID-19

Abstract 44: Childhood Predictors of Type 2 Diabetes in Young Adulthood to Middle-age: The International Childhood Cardiovascular Cohort (i3c) Consortium

Introduction: Although childhood obesity is related to incident adult type 2 diabetes (T2DM), whether other childhood risk factors relate to T2DM is not clear and no childhood risk score has been established to help with prediction of T2DM. Methods: We examined childhood risk factors in The International Childhood Cardiovascular Cohort (i3C) Consortium with a goal of developing a risk score for predicting adult T2DM. The pooled datasets included childhood measurements during the 1970s to 1990s. Participants were re-contacted at mean age 40 yr and completed a health questionnaire including self-report of adult T2DM (occurrence age, medication use). Results: N = 4,425 i3C participants with measurements during ages 3-19 yr. 225 (5.1%) reported onset of T2DM between ages 20-55 (mean 38.4) years (82% reported use of a valid antidiabetic medication). After stepwise regression, age-sex standardized deviates (mean of all repeated measures during ages 3-19 yr) of body mass index (BMI), glucose and ln(insulin) were associated with incident T2DM; the unadjusted risk curves visibly steepened at about 0.75 standard deviates (Figure, top panel). In multivariable models, BMI, glucose and ln(insulin) in a single model were associated with adult T2DM after adjustment for sex, race, cohort, and individual mean childhood age and calendar year. Prediction of incident T2DM was directly related to the number of the three risk factors with levels above +0.75 standard deviates (0: 3.2% (96/2966), 1: 6.2% (63/1,012), 2: 14.3% (56/392), 3:18.2% (10/55); the corresponding adjusted HR (95%CI) for the counts were 1 (reference), 1.8 (1.3, 2.4), 4.7 (3.3, 6.5), and 6.7 (3.5, 13.1), respectively). The +0.75 standard deviates in natural units are given in the Figure (bottom panel), e.g. about 92 mg/dL for glucose at age 12-14. Conclusion: The study suggests that a childhood risk score composed of BMI, glucose and insulin is a strong predictor of adult T2DM. Provisionally suggested cutpoints are lower than currently defined normal.

Enhancing the Efficacy of Stem Cell Therapy with Glycosaminoglycans.

SummaryHuman mesenchymal stem cell (hMSC) therapy offers significant potential for osteochondral regeneration. Such applications require their ex vivo expansion in media frequently supplemented with fibroblast growth factor 2 (FGF2). Particular heparan sulfate (HS) fractions stabilize FGF2-FGF receptor complexes. We show that an FGF2-binding HS variant (HS8) accelerates the expansion of freshly isolated bone marrow hMSCs without compromising their naivety. Importantly, the repair of osteochondral defects in both rats and pigs is improved after treatment with HS8-supplemented hMSCs (MSCHS8), when assessed histologically, biomechanically, or by MRI. Thus, supplementing hMSC culture media with an HS variant that targets endogenously produced FGF2 allows the elimination of exogenous growth factors that may adversely affect their therapeutic potency.

The Pediatric Acute Leukemia Fusion Oncogene ETO2‐GLIS2 Increases Self‐Renewal and Alters Differentiation in a Human Induced Pluripotent Stem Cells‐Derived Model

Supplemental Digital Content is available in the text.

WASP Restricts Active Rac to Maintain Cells' Front-Rear Polarization.

SummaryEfficient motility requires polarized cells, with pseudopods at the front and a retracting rear. Polarization is maintained by restricting the pseudopod catalyst, active Rac, to the front. Here, we show that the actin nucleation-promoting factor Wiskott-Aldrich syndrome protein (WASP) contributes to maintenance of front-rear polarity by controlling localization and cellular levels of active Rac. Dictyostelium cells lacking WASP inappropriately activate Rac at the rear, which affects their polarity and speed. WASP’s Cdc42 and Rac interacting binding (“CRIB”) motif has been thought to be essential for its activation. However, we show that the CRIB motif’s biological role is unexpectedly complex. WASP CRIB mutants are no longer able to restrict Rac activity to the front, and cannot generate new pseudopods when SCAR/WAVE is absent. Overall levels of Rac activity also increase when WASP is unable to bind to Rac. However, WASP without a functional CRIB domain localizes normally at clathrin pits during endocytosis, and activates Arp2/3 complex. Similarly, chemical inhibition of Rac does not affect WASP localization or activation at sites of endocytosis. Thus, the interaction between small GTPases and WASP is more complex than previously thought—Rac regulates a subset of WASP functions, but WASP reciprocally restricts active Rac through its CRIB motif.

The Molecular Basis of Long First Remissions in Normal Karyotype AML Patients

Currently, it is not clear why some patients with acute myeloid leukemia (AML) can be "cured" with chemotherapy alone; are they living with small amounts of disease that is held in check by immunologic (or other) mechanisms, or is their disease really eradicated? The percentage of cytogenetically normal AML patients who have long (>5 years) first remissions (LFRs) after chemotherapy alone is low (about 9.1% in patients <60 years and 1.6% in >60 years1). For this reason, most intermediate risk patients are offered allogeneic transplantation to decrease their risk for relapse. To better understand mechanisms of chemotherapy sensitivity in AML, we performed an analysis of the mutation landscape and persistence, using samples from 8 normal karyotype LFR patients (without CEBPA mutations) who received standard "7+3" induction and high dose cytarabine consolidation as their only therapy. The mean age at diagnosis was 43.5 years, and the mean follow up in first remission is 7.6 years; none of these patients has relapsed to date. For each case, we performed enhanced exome sequencing at diagnosis (235x coverage of the entire exome, and ~1008x coverage of recurrently mutated AML genes). Each case had at least one documented AML driver mutation, with a median of 29 somatic mutations in the exome space. We created probes for 225 mutations (mean 28 per case), and performed error-corrected sequencing (Haloplex) for all available remission samples. The mean depth of Haloplex coverage was 1607x, and each sample had at least one AML-specific mutation assayed, with a sensitivity of 1 cell in 1,750 (0.06%). 7/8 patients demonstrated complete clearance of all mutations in all remission samples tested, which was confirmed with digital droplet PCR for 5 cases, with a sensitivity of detection of 1 cell in 100,000. In one case, we detected a persistent ancestral clone harboring DNMT3AR882H, which can be associated with long first remissions for some patients2. Strikingly, the founding clone in all 8 cases had one or more somatic mutations in genes known to drive cell proliferation (e.g. MYC, FLT3, NRAS, PTPN11, Figure 1 top panel). These are usually subclonal mutations that occur late during leukemic progression, suggesting that the presence of a "proliferative hit" in the founding clone might be important for chemotherapy clearance of all the AML cells in a given patient. To support this hypothesis, we analyzed the mutational clearance of 82 AML cases with paired diagnosis and day 30 post-chemotherapy bone marrow samples. We observed that, whether present in the founding clone or in subclones, mutations in MYC, CEBPA, FLT3, NRAS, and PTPN11 cleared after induction chemotherapy in all samples, while other mutations were often persistent at day 30 (e.g. DNMT3A, IDH1, IDH2, NPM1, TET2; Figure 1 bottom panel). Compared to other published sequencing studies of AML, MYC and NRAS mutations were significantly enriched in this small cohort (MYC p= 0.002, and NRAS p= 0.034), with MYC enrichment being particularly striking (37.5% versus 1.8%). All MYC mutations were canonical single base substitutions occurring in the highly conserved MYC Box 2 domain at the N-terminus of MYC (p.P74Q or p.T73N). Overexpression of MYCP74Q in murine hematopoietic progenitors prolonged MYC half life (89 min vs. 44 min for wild type), and enhanced cytarabine sensitivity at all concentrations tested (range 10-1000 nM, p=0.0003), both in vitro and in a MYC-driven leukemia model in vivo. MYC expression measured with flow cytometry in the blasts of the LFR samples was significantly higher (p=0.045) compared to unfavorable risk (complex karyotype) or other intermediate risk categories, but similar to good risk AML (biallelic CEBPA mutations, core binding factor fusion-associated AML, and AML with isolated NPMc), suggesting that activation of the MYC pathway may represent a shared feature of chemosensitive patients. Taken together, these data suggest that some intermediate patients who are effectively "cured" with chemotherapy alone may not have persistent subclinical disease, nor retained ancestral clones that could potentially contribute to relapse. Importantly, these patients often have mutations driving cell proliferation in the founding clone, indicating that the presence of specific mutations in all malignant cells may be critical for complete AML cell clearance with chemotherapy. 1. Blood Adv. 2018 Jul 10; 2(13): 1645-1650 2. N Engl J Med 2018; 378:1189-1199 Disclosures No relevant conflicts of interest to declare.

In Vivo Assessment of Intracellular Dynamics Comparing Injection Versus Oral Azacitidine in a Phase IIb Investigator Initiated Clinical Trial

In Vivo Assessment of Intracellular Dynamics Comparing Injection Versus Oral Azacitidine in a Phase IIb Investigator Initiated Clinical Trial

Additional Cytotoxic Chemotherapy Is Unlikely to Eliminate Measurable Residual Acute Myeloid Leukemia (AML)

Additional Cytotoxic Chemotherapy Is Unlikely to Eliminate Measurable Residual Acute Myeloid Leukemia (AML)

Sound absorption of face-centered cubic sandwich structure with micro-perforations

This paper presents a novel ultra-lightweight micro-perforated sandwich structure with face-centered cubic (FCC) core as a sound absorber, which encompasses excellent mechanical features as well as acoustic properties. The structure, which consists of air cavities and small perforations on both the top panel and the corrugated plates, is considered as a combination of multi-layered micro-perforated panel absorbers (MPPAs). A theoretical model that makes use of an equivalent electrical circuit (EEC) approach is established. Validation of the model with experimental results and numerical simulations shows good agreement. The dependence of the acoustic absorption on features of the perforations and of the FCC structure is discussed. Based on the observations, an optimized structure is proposed by using simulated annealing method to yield good acoustic absorption property in the low frequency range.

Editor's Note: Estrogen Receptor α Promotes Breast Cancer by Reprogramming Choline Metabolism.

The editors are publishing this note to alert readers to concerns about [this article][1] ([1][2]). The editors were made aware of duplicated Western blot bands in Fig. 4A. Specifically, two sets of Western blot bands are duplicates: (i) T47D CCTα in the top right panel and T47D cadherin in the

P4574Marked changes in the incidence of subendocardial and ST elevation myocardial infarctions: a twenty-one year study

Abstract Background/Introduction The use of new biomarkers, pharmacologic and interventional therapies have resulted in marked changes of the type of myocardial infarction. Purpose The purpose of this study is to report on the changes in the incidence of subendocardial and ST elevation myocardial infarctions (STEMI) in the state of New Jersey from 1994 to 2015 and describe possible explanations for this secular change. Methods The rate of occurrence of ST elevation myocardial infarction (ICD9 410.00) and of subendocardial (NSTEMI, ICD9 410.70 to 410.72) myocardial infarction was studied using the Myocardial Infarction Data Acquisition System (MIDAS), a statewide database of all admissions for acute myocardial infarction in New Jersey with more than 30 years longitudinal follow up. Admissions for myocardial infarction and unstable angina as well as for conditions associated with troponemia (heart failure ICD9 418.XX, chronic kidney disease ICD9 584.XX, 585.XX, 403.XX, diabetes ICD9 250.XX) were also identified. Associations among variables were examined using logistic regression and linear error in variables (LEIV) models. Results During the time period under consideration, there was a decrease in the total number of MIs in New Jersey by 17.5%, the number of STEMIs decreased by 67.4%, while the rate of NSTEMIs increased by 85.8% (top panel). There was a complementary relationship between the annual number of admissions for unstable angina to the annual number of admissions for NSTEMIs (bottom panel). The effect was more pronounced between 1994 and 2002 and plateaued thereafter corresponding to widespread use of troponins in the diagnosis of acute coronary syndromes. Conclusions The marked change in the type of myocardial infarction was due in part to the use of troponins that resulted in the classification of patients with unstable angina as patients with subendocardial infarction. These data support regionalization of facilities specializing in immediate intervention in STEMI patients.

3225Global longitudinal strain measured using feature-tracking cardiac magnetic resonance imaging is an independent predictor of all cause mortality in patients with preserved ejection fraction

Abstract Introduction Ejection fraction is the principal measure used clinically to assess cardiac mechanics and provides significant prognostic information. However, echocardiographic strain imaging has shown significant abnormalities of myocardial deformation can be present despite preserved ejection fraction, which maybe associated with adverse prognosis. Cardiac-Magnetic-Resonance (CMR) feature-tracking techniques now allow assessment of strain from routine cine-images, without specialized pulse sequences. Whether abnormalities of strain measured using CMR feature-tracking have prognostic value in patients with preserved ejection fraction is unknown. Purpose To evaluate the prognostic value of CMR feature-tracking derived global longitudinal strain (GLS) in a large multicenter population of patients with preserved ejection fraction. Methods Consecutive patients with preserved ejection fraction (EF ≥50%) and a clinical indication for CMR at four US medical centers were included in this study. Feature-tracking GLS was calculated from 3 long-axis-cine-views. The primary endpoint was all-cause death. Cox proportional hazards regression modeling was used to examine the independent association between GLS and death. The incremental prognostic value of GLS was assessed in nested models. Results Of the 1274 patients in this study, 115 died during a median follow-up of 6.2 years. By Kaplan-Meier analysis, patients with GLS ≥ median (−20%) had significantly reduced event free survival compared to those with GLS < median (log-rank p<0.001) (Figure, top panel). The continuous relationship between GLS and the hazard of death is shown in the cubic spline (Figure, lower panel). By Cox multivariable regression modeling, each 1% worsening in GLS was associated with a 23.6% increased risk-of-death after adjustment for clinical and imaging risk factors (HR=1.236 per %; p<0.001). Addition of GLS in this model resulted in significant-improvement in the global-chi-square (67 to 168; p<0.0001) and Harrel's C-statistic (0.716 to 0.825; p<0.0001). Conclusions CMR feature-tracking derived GLS is a powerful independent predictor of mortality in patients with preserved ejection fraction, incremental to common clinical and imaging risk factors.

Seismic Performance of Precast Shear Wall–Diaphragm Connection: A Comparative Study with Monolithic Connection

The primary objective of the present study is to understand the behaviour of innovative precast shear wall–slab connection subjected to reverse cyclic loading. The response of the precast connection is then compared with that of the similar monolithic connection. One-third scaled-down models were used for the investigation. In the monolithic connection, typically used U-shaped bars were employed for the connectivity between shear wall and slab. In the precast connection, the connectivity was established in two steps. In the first step, the precast slab and a lower half portion of the shear wall were connected through dowel bars and tied with the screed reinforcement. In the second step, the bottom and top panel of the shear walls were connected through the dowel bars. The specimens were subjected to reverse cyclic loading at the slab ends on both sides. The seismic performance regarding strength, load–displacement relationship, crack pattern, energy dissipation, moment–rotation curve, stiffness and ductility were obtained and compared with the monolithic connection. The average ultimate load and moment-carrying capacity of the precast specimen are found to be 28.66% and 21.74% greater than the monolithic specimen. The cumulative energy dissipation and ductility factor of the precast specimen are found to be 128.95% and 74.34% higher than the monolithic specimen. It is concluded that the precast specimen showed better performance concerning the ultimate load, moment, energy dissipation and ductility with reference to the monolithic specimen.

The Intergenerational Impact of China's New Rural Pension Scheme

The Intergenerational Impact of China's New Rural Pension Scheme

Correction: The ARF exchange factors Gea1p and Gea2p regulate Golgi structure and function in yeast.

The authors would like to offer clarifications for J. Cell Sci. (2001) 114, 2241-2253.A reader informed the journal that the western blots in Fig. 4D had several splices. The corresponding author, Catherine Jackson, was able to provide the full original blots for Fig. 4D, which supported the data shown. The journal decided that no correction for this figure is required as the paper was published before the policy on blot presentation was implemented.During re-analysis of the original data for this paper, the authors found that images of several cells were spliced in Fig. 6A,B. The authors state:“We report in the main text the percentages of cells with different phenotypes for each of the conditions shown in Fig. 6, and present only one class of phenotype for each condition in the figure, making it clear that the cells shown are not representative of the entire population of cells when more than one phenotype is present (Fig. 6A,B, bottom panels). It would have been sufficient to show only one cell per condition (as we do for Fig. 6B, top panel), a frequent practice at the time. However, for Fig. 6A top panel and Fig. 6B, we chose 2–3 cells and grouped them together, a mode of presentation that can be found in other contemporaneous papers. Although the frequency of each phenotype is provided in the main text, we realized that for a reader looking at the figure in isolation, this fact would not have been clear. Hence, in light of the original data, we amended Fig. 6 to show the edges of all the cropped images.”The revised figure and legend are shown here. The authors apologise to readers for this presentation error, which does not impact the results or conclusions of the paper.

Individual and combined presenilin 1 and 2 knockouts reveal that both have highly overlapping functions in HEK293T cells

Presenilins 1 and 2 (PS1 and 2) are the catalytic subunits of γ-secretase, a multiprotein protease that cleaves amyloid protein precursor and other type I transmembrane proteins. Previous studies with mouse models or cells have indicated differences in PS1 and PS2 functions. We have recently reported that clinical γ-secretase inhibitors (GSIs), initially developed to manage Alzheimer's disease and now being considered for other therapeutic interventions, are both pharmacologically and functionally distinct. Here, using CRISPR/Cas9-based gene editing, we established human HEK 293T cell lines in which endogenous PS1, PS2, or both have been knocked out. Using these knockout lines to examine differences in PS1- and PS2-mediated cleavage events, we confirmed that PS2 generates more intracellular β-amyloid than does PS1. Moreover, we observed subtle differences in PS1- and PS2-mediated cleavages of select substrates. In exploring the question of whether differences in activity among clinical GSIs could be attributed to differential inhibition of PS1 or PS2, we noted that select GSIs inhibit PS1 and PS2 activities on specific substrates with slightly different potencies. We also found that endoproteolysis of select PS1 FAD-linked variants in human cells is more efficient than what has been previously reported for mouse cell lines. Overall, these results obtained with HEK293T cells suggest that selective PS1 or PS2 inhibition by a given GSI does not explain the previously observed differences in functional and pharmacological properties among various GSIs.

Factors Associated With Mortality and Response to Extracorporeal Photopheresis in Lung Allograft Recipients With Bronchiolitis Obliterans Syndrome.

This study was designed to identify factors associated with clinical response to extracorporeal photopheresis (ECP) and mortality after ECP in lung allograft recipients with bronchiolitis obliterans. Forced expiratory volume in 1 second (FEV1) values obtained 6 months before (baseline) and 6 months after initiation of ECP were used to plot the linear relationship between FEV1 versus time before and after ECP. Response to ECP was assigned when a positive integer was derived after subtracting the baseline rate of decline from the rate of decline 6 months after ECP. Univariate and multivariate logistic regression analyses were used to identify demographic, treatment-related factors or spirometric parameters that may be associated with response to ECP or mortality at either 6 or 16 months after initiation of ECP. Forced expiratory volume in 1 second just before ECP was associated with mortality (P = 0.007) at 16 months after ECP initiation. An FEV1 of 1.50 L or less had a sensitivity of 87% and specificity of 60% to identify patients who died within 16 months after ECP initiation. Patients whose FEV1 decline exceeded 40 mL/month were 12 times more likely to have a response to ECP (P = 0.0001). Patients whose decline in FEV1 before ECP was statistically significant (P < 0.05) were nearly 10 times (P = 0.008) more likely to respond to ECP. Forced expiratory volume in 1 second is an important predictor of mortality, and the response to ECP is influenced by both the extent (>40 mL/mo) and statistical significance of the relationship between FEV1 versus time before ECP initiation. Therefore, earlier bronchiolitis obliterans detection and more timely implementation of ECP (ie, when FEV1 values >1.5 L) should be considered especially in patients with a more aggressive rate of decline of lung function.