Citations

Synchrotron based hydroxyl radical footprinting (HRF) was employed to study the formation of Aβ 42 oligomers, believed to be the neurotoxic species in Alzheimer's disease. Rates of oxidative modification for seventeen individual residues (F4, Y10, H13, H14, L17, F19, F20, V24, K28, I31, I32, M35, V36, V39, V40, I41) were determined for monomer, 2‐4mer, and oligomer Aβ 42 relative to intrinsic rates of modification determined for the same residues in fragment peptide. The ratio of measured and intrinsic rates reveals protection from hydroxyl radical attack due to reduced side chain solvent accessibility for structured Aβ 42 . Protection observed in the intrinsically disordered Aβ 42 monomer is consistent with transient C‐terminal β‐strand formation and partial folding in the hydrophobic core region. Our results also reveal dimerization (and further assembly) contacts that are consistent both with oligomer C‐terminal antiparallel β‐sheet formation previously indicated from published solid state NMR work studies and newly detected N‐terminal structure.

Background Esophageal pathology such as perforation or malignancy often necessitates esophageal reconstruction. However, the optimal method of esophageal primary repair and anastomosis are unclear. We aim to determine stress to failure of esophageal anastomosis for full thickness and mucosal‐submucosal anastomotic techniques. Methods Six esophagi from freshly sacrificed 60 kilogram swine were transected at the mid thoracic portion of the esophagus. Esophageal anastomoses were created and subsequently primarily anastomosed using 2‐0 running continuous polyglactin (Vicryl, Ethicon, Somerville, NJ) suture either as full thickness or partial thickness (mucosa‐submucosa alone). The sutured specimens were then mounted on cannulae. Using a syringe pump system, the esophagi were infused with colored saline while monitoring pressure continuously with optical tracking for detection of fluid leak. Data acquisition software was used to collect stress‐to‐failure data. Synchronized video and pressure data were analyzed post hoc for visualization of the leak and concurrent pressure to determine failure pressure. Results The full‐thickness anastomosis failed at a mean ± (SEM) pressure of 15.33 ± (1.45) mmHg while the mucosal‐submucosal anastomosis failed at a mean ± (SEM) pressure of 36.33 ± (3.76) mmHg, ( P = 0.02, unpaired t ‐test with Welch's correction). MATLAB (MathWorks, Natick, MA) was used calculate the statistical results. Conclusion While the optimal in vivo method for esophageal anastomosis is unclear, we present evidence that an anastomosis incorporating only the mucosa and submucosa may be superior to full thickness. Additional research is needed to further clarify optimal technique and post‐operative outcomes. Support or Funding Information Dr. Aho reports funding from a Grant from the National Heart, Lung, and Blood Institute T32 HL105355, a component of the National Institutes of Health (NIH).

Background Obesity‐associated metabolic disorders have been shown to be related to body fat distribution. Specifically, increases in visceral fat are more detrimental than increases in subcutaneous fat depots. However, the underlying mechanisms through which visceral fat potentiates cardiometabolic dysregulation are not completely understood. In recent years, the presence of localized renin angiotensin system (RAS) in adipose tissue and its role in regulating blood pressure and metabolism has been recognized. Therefore, we investigated if differential expression of RAS components in visceral versus subcutaneous adipose tissue may help explain pathological effects of visceral fat. Hypothesis We hypothesized that compared to subcutaneous adipose tissue, visceral adipose tissue has increased expression of RAS proteins involved in the pro‐inflammatory and pro‐fibrotic ANGII‐AT1R pathway but attenuated expression of proteins mediating the anti‐inflammatory and anti‐fibrotic ANG1‐7‐MASR pathway. Methods We determined the expression of RAS proteins in paired subcutaneous and visceral (omental) adipose tissue samples from obese individuals undergoing surgery (n = 30, BMI: 46.1 ± 7.3 kg/m 2 , age: 46.5 ± 14.4 years) by Western blot. We examined proteins involved in 1) Ang II generation: angiotensinogen (AGT), angiotensin converting enzyme (ACE), chymase; 2) Ang1‐7 generation: ACE‐2; and 3) cellular receptors: angiotensin I receptor (AT1R), MAS receptor (MASR), and aldosterone receptor. Results Comparison of protein expression between subcutaneous and visceral adipose tissue samples showed no changes in proteins involved in generation of angiotensin peptides, namely AGT (0.64 ± 0.57 vs. 0.33 ± 0.28, p = 0.11, n = 12), chymase (0.27 ± 0.23 vs. 0.36 ± 0.41, p = 0.77, n = 22), and ACE‐2 (0.23 ± 0.41 vs. 0.08 ± 0.09, p = 0.21, n = 15). However, ACE expression tended to be higher in subcutaneous vs. visceral fat (0.10 ± 0.06 vs. 0.06 ± 0.03, p = 0.052, n = 12). Furthermore, expression of cellular receptor AT1R was higher in subcutaneous fat (0.24 ± 0.34 vs. 0.07 ± 0.09, p = 0.03, n = 12), and MASR was lower in subcutaneous fat (0.22 ± 0.26 vs. 0.88 ± 0.99, p = 0.01, n = 20). Aldosterone receptor expression (0.21 ± 0.22 vs. 0.14 ± 0.13, p = 0.52, n = 11) was not altered in subcutaneous and visceral depots. Conclusions Proteins involved in the generation of angiotensin peptides are not differentially expressed in subcutaneous versus visceral fat depots. However, expression of cellular receptors is different such that inflammatory and fibrotic effects of RAS may be more predominant in subcutaneous fat. Therefore, RAS is likely to play a role in limiting subcutaneous fat expansion thereby leading to increased visceral fat depots. Furthermore, our findings do not support the role of visceral RAS in development and progression of cardiovascular pathology associated with increased visceral fat depots. Support or Funding Information MAP and KRS were supported by the American Physiological Society Stride Summer Undergraduate Research Fellowship NHLBI 1 R25 HL115473‐01.

Perceived self‐efficacy influences how one approaches goals, tasks, and challenges, and has been shown to affect motivation and academic behaviors. The ability to accurately assess one's competency and limitations is also a critical skill for physicians. Research has shown that medical students can underestimate their perceived clinical skills compared to observer assessment, but limited research has addressed perceived knowledge specific to anatomy. This study compares student perception and faculty evaluation of academic competency in a gross anatomy course. Additionally, this study revisits whether daily audience response system (ARS) questions is a predictor of academic success, and the impact of near‐peer tutoring on student performance. Data from 51 first year medical students in a 7‐week gross anatomy course were evaluated. At the end of week 3 of the course, students with a cumulate ARS grade below the 70% pass requirement were contacted by faculty with individualized formative feedback on academic performance and presented with opportunities for improvement and formal tutoring. All students irrespective of performance were made aware of opportunities for tutoring. Six near‐peer teaching assistants tutored students who requested help. Mean ARS scores and laboratory practice practical grades were evaluated before and after the students received formative feedback. Students were placed into one of four groups based on two factors: (1) their perceived ability to succeed in the course, and (2) faculty perception of their potential for success. Students in groups 1 and 3 were identified by faculty members as potentially at risk, whereas groups 2 and 4 were viewed as low risk. From the students' perspective, those in groups 1 and 2 self‐identified as needing assistance, whereas groups 3 and 4 felt that they were not at risk of failing the course. Of the 12 students contacted by faculty, 6 (12% of the class) requested a tutor (group 1). Additionally, 11 (21%) students who were not contacted by faculty requested a tutor (group 2). Six (12%) students that were contacted by faculty did not request a tutor (group 3), while 28 (55%) were not contacted by faculty and did not request a tutor (group 4). The averages of daily ARS scores between groups after feedback was significantly greater in group 4 compared to groups 1 and 2 (68% vs 61% and 63%, p=0.04 and 0.04, respectively). In addition, the post faculty feedback practical average scores were significantly higher in group 4 compared to group 1 (67% vs 56%, p=0.04). These data show that in an environment that fosters acceptance of academic support, students are more receptive to receiving and reaching out for help from near‐peer tutors. Formative feedback from ARS scores continue to be a reliable indicator for tracking student performance. Improvement of student performance in final course evaluation may be a positive indicator of success from near‐peer tutoring. Support or Funding Information None

Airway inflammation is a key aspect of diseases such as asthma. Several inflammatory cytokines (e.g., TNFα and IL‐13) enhance cytosolic Ca 2+ concentration ([Ca 2+ ] cyto ) triggering airway smooth muscle (ASM) hyperreactivity (enhanced contractile state). Airway inflammation also induces ASM proliferation and remodeling (enhanced ASM synthetic state). In various diseases inflammation induces sarcoplasmic/endoplasmic reticulum (SR/ER) stress, which decreases SR/ER‐mitochondria interactions, with downstream effects on Ca 2+ regulation. In vascular smooth muscle cells, SR/ER stress promotes smooth muscle contraction and proliferation by disrupting SR/ER‐mitochondrial coupling. We hypothesized that inflammation induces SR/ER stress in human ASM, thereby altering expression of mitochondrial fission/fusion proteins (dynamin‐related protein ‐ Drp1 and mitofusin ‐ Mfn2) with downstream impact on ASM Ca 2+ regulation and contractility as well as proliferation. Human ASM cells were isolated from lung specimen incidental to patient surgery and exposed to TNFα or IL‐13 for 24h. Expression of SR/ER stress markers including PERK (protein kinase‐like endoplasmic reticulum kinase), Bip (Binding immunoglobulin protein also known as 78 kDa glucose‐regulated protein), PDI (protein disulfide isomerase), and mitochondrial fission/fusion proteins Drp1 and Mfn2 were evaluated by Western blot. The expression of the spliced isoform of the transcription factor XBP1 was investigated by quantitative real‐time PCR (RT‐PCR). For mitochondrial morphology (fission/fusion), ASM cells were loaded with Mitotracker Green and images analyzed using ImageJ and Matlab to quantify mitochondrial Form Factor and Aspect Ratio. The effect of inflammation induced SR/ER stress on human ASM proliferation was evaluated using CyQUANT assay. We found that after 24 h exposure to TNFα and IL‐13: 1) expression of SR/ER stress protein markers (PERK, BiP and XBP1) increased; 2) Drp1 expression increased and Mfn2 expression decreased; 3) mitochondrial fragmentation increased; 4) [Ca 2+ ] cyt and [Ca 2+ ] mito responses to ACh stimulation were uncoupled; 5) net [Ca 2+ ] cyt increased; 6) contractility increased; and 7) ASM cell proliferation increased. Together these results suggest that inflammation induces SR/ER stress that leads to changes in Mfn2 and Drp1 expression, which alter mitochondrial dynamics and Ca 2+ regulation resulting in ASM hyperreactivity and remodeling. Support or Funding Information NIH grant HL126451 (GCS).

Background Much research has been done into finding specific molecular pathways involved in the metastatic and invasive potential of breast cancer cells. One of the major discoveries of this past decade has been microRNAs; which are single‐stranded non‐coding RNA molecules. Interestingly a single microRNA can influence the expression of hundreds of proteins, thereby highlighting their significance as modulators of gene expression. Apart from their role in regulating various intracellular processes, abnormal changes in microRNA expression have been linked to various human cancers. Until recently the nature and impact of most of these changes in breast cancer were unclear. However, some of the microRNA dysregulations have now been correlated with clinic‐pathological features such as Her2, ER and PR status, tumor stage, angiogenesis, metastasis and invasion. Of note to our study, compiling data has shown that microRNAs regulate cancer metastasis by influencing multiple steps in the metastatic cascade. This review will focus on key microRNAs associated with breast cancer invasion and metastasis; recently referred to as ‘metastamirs’. Methods We used the program EndNote X7 to research PubMed published articles regarding microRNAs in Breast Cancer. The terms “miRNAs” and “Breast cancer” were used as search words and the publication years were specified to range from 2005 to 2016. Our preliminary search revealed no articles pertaining to our topic before the year 2005, hence our paper covers nearly the entire literature on the topic. The retrieved records were then reviewed, and all those pertaining to metastasis and invasion in breast cancer were included in our study. Results Our EndNote search retrieved a total of 2820 abstracts, of which more than 280 were pertaining to metastasis and invasion in breast cancer. These abstracts were then read thoroughly, and roughly 130 metastasis/invasion specific microRNAs or clusters of microRNAs were identified. Interestingly, some microRNAs were shown to promote metastasis and invasion when up‐regulated, while others did so when they were down‐regulated. Nonetheless, all the identified microRNAs brought about their effects through various molecular pathways, some at more critical stages in the metastasis cascade than others. presents some of the commonly identified pro‐metastatic & anti‐metastatic microRNAs. Conclusion The last decade has been revelationary with regards to non‐coding RNAs, microRNAs, and their association with various cancers. Our comprehensive review highlights the various microRNAs involved in key pathways within the breast cancer metastatic pathway. Co‐relating these microRNAs to clinically targetable pathophysiological points could revolutionize our fight against breast cancer; by not only serving as diagnostic & prognostic markers, but also being potential therapeutic targets. Carefully designed translational studies seem to be the call of the hour, to finally give us the much‐needed edge over breast cancer. Support or Funding Information The authors report no source of funding or support.

Introduction The contribution of metabolic vs mechanical stimuli related to the group III/IV afferent‐mediated exercise pressor reflex (EPR) remains incompletely understood. High pedal frequency during cycling increases type II muscle fiber recruitment and may contribute to increased anaerobically‐mediated metabolic byproduct accumulation. This may lead to an augmented rise in systolic blood pressure (SBP) and, hence, EPR in humans. Thus, we hypothesized, 1) Mean arterial pressure (MAP) will be increased during exercise with SubRCO vs without NoRCO irrespective of pedal frequency, and 2) MAP will be augmented further during exercise with SubRCO at high pedal frequencies. Methods Healthy adults (N=13; 46% men; age 36±19 yr; BMI 24±4 kg/m 2 ) participated in 2 study visits, each consisting of 2 sessions of fixed load (20 W) recumbent cycle ergometry with bilateral upper thigh pressure tourniquets for 8 min at a fixed pedal frequency of 35 (LOW) or 100 (HIGH) rpm (randomized). Cuffs remained at 0 mm Hg for the first 3 min (CTL), and were then randomized to 0 mm Hg (NoRCO) or 90 mm Hg (SubRCO) for the final 5 min of exercise. SBP and diastolic blood pressure (DBP) were measured via manual sphygmomanometry at the end of CTL and end‐exercise (END). MAP was calculated as (SBP + 2 * DBP)/3. Results There were no between or within condition differences at CTL for MAP (P>0.05). In contrast, for between SubRCO and NoRCO differences at END, MAP was increased in SubRCO vs NoRCO in both HIGH (106±12 vs 92±13 mm Hg, respectively) and LOW (94±9 vs 85±8 mm Hg, respectively) (P<0.05). At END, MAP was increased in HIGH vs LOW within both SubRCO and NoRCO (P<0.01). Within both NoRCO (129 ± 18 vs 117 ± 11 mm Hg) and SubRCO (125 ± 15 vs 113 ± 10 mm Hg) at CTL, SBP was increased in HIGH vs LOW, respectively (P<0.01). At CTL, there were no between SBP NoRCO vs SubRCO differences in HIGH or LOW. Within SBP differences for NoRCO (134 ± 19 vs 113 ± 11 mm Hg) and SubRCO (147 ± 20 vs 127 ± 12 mm Hg) in HIGH vs LOW persisted to END, respectively (P<0.01). SBP increased at END vs CTL within SubRCO for both HIGH and LOW (P<0.02), but not within NoRCO (P>0.05). Lastly, SBP increased in SubRCO vs NoRCO at END in both HIGH and LOW (both P<0.01). There were no differences for DBP at any time point (P>0.05). Conclusion These data support our hypothesis that, independent of pedal frequency, SubRCO provokes increased MAP during fixed‐load submaximal exercise as a function of increased SBP. However, high pedal rate accompanied by SubRCO results in exaggerated SBP‐mediated increased MAP. These data contribute to an improved understanding of the stimuli that activate group III/IV afferents during exercise in humans. Support or Funding Information NHLBI RO1 HL126638; AHA 16POST30260021

Introduction The exercise pressor reflex (EPR) is suggested to be linked to activated group III/IV afferents. However, the predominating mechanical and/or metabolic stimuli by which the EPR is mediated remains unclear. It is suggested that high pedal frequency increases the recruitment of type II muscle fibers, which may result in augmented production of metabolic byproducts as a function of increased anaerobic processes. Therefore, this study tested the hypothesis that compared to low frequency, high pedal frequency will be associated with increased blood pressure during postexercise suprasystolic regional circulatory occlusion (SupraRCO). Further, although subsystolic RCO (SubRCO) during exercise is suggested to provoke venous distension associated with mechanically‐mediated activation of group III/IV afferents, we hypothesized the rise in blood pressure during postexercise SubRCO will be blunted vs postexercise SupraRCO irrespective of pedal frequency. Methods Healthy adults (N=13, 6 male, 7 female), age 36±19 yrs participated in 2 days of testing, each consisting of 3 sessions of recumbent cycle ergometry at pedal frequencies of 100 (HIGH) or 35 (LOW) rpm (randomized) at 20 W for 8 min. At cessation of exercise, bilateral upper thigh pressure tourniquets were inflated to SupraRCO (200 mm Hg), SubRCO (90 mm Hg), or no inflation (NR) for 2 min (randomized). Blood pressure via manual sphygmomanometry was measured at rest, end‐exercise, and 2 min postexercise. Mean arterial pressure (MAP) was calculated as (SBP + 2 * DBP)/3. Results For both rest and end‐exercise, MAP did not differ across repeated transients for either HIGH or LOW (both P>0.05). However, mean MAP across repeated transients was greater in HIGH vs LOW at end‐exercise (P<0.05). At 2 min postexercise in HIGH, compared to NR (89±8 mm Hg), MAP was higher in SupraRCO (105±18 mm Hg; P=0.02), but not in SubRCO (98±9 mm Hg; P=0.38); whereas MAP did not differ between SupraRCO and SubRCO (P=0.70). Similarly, at 2 min postexercise in LOW, MAP was increased in SupraRCO vs NR (96±10 vs 85±7 mm Hg, respectively; P=0.02); whereas there were no differences between SubRCO (88±7 mm Hg) with either NR (P=0.91) or SupraRCO (P=0.23). Lastly, compared to LOW, MAP was greater in HIGH at 2 min postexercise for both SupraRCO (P<0.01) and SubRCO (P<0.01), but not in NR (P=0.59). Conclusions In intact exercising humans it remains challenging to elucidate exact contributions of group III/IV afferents to the EPR. However, these data are consistent with the role of group III/IV afferents in contributing to the EPR in humans. New to this field, our observations suggest that at low fixed workloads, increased pedal frequency augments the postexercise pressor response to both SupraRCO and SubRCO in healthy adults. These data contribute to an improved understanding of specific stimuli associated with the EPR linked to group III/IV afferents in humans. Support or Funding Information NHLBI R25 HL115473; NHLBI RO1 HL126638; AHA 16POST30260021

Introduction Additional oxygen uptake (VO 2 ) beyond muscle oxygen (O 2 ) utilization rates may coincide with exercise at or above the lactate threshold, which may be illustrated using a Phase II to III two‐exponential on‐transient VO 2 kinetics model. This is expected to prolong mean response time (MRT) to VO 2 steady‐state commensurate with increased O 2 deficit (O 2 def), which suggests reduced energy efficiency. Several factors may provoke metabolic acidosis during ergometry including exaggerated pedal frequency related to greater recruitment of type II muscle fibers. Also unclear is whether locomotor subsystolic regional circulatory occlusion (SubRCO), used to study group III/IV afferents during ergometry, accelerates metabolic acidosis due to influences on arterial‐to‐venous gradients. Thus, this study aimed to test the hypothesis that the transition from exercise without SubRCO to with SubRCO during fixed high rate pedaling leads to a Phase III rise in on‐transient VO 2 kinetics and, hence, augmented MRT and O 2 def during hyperbolic submaximal exercise. Methods Healthy adults (N = 15, 53% male; age 36 ± 17 years; BMI 24 ± 4 kg/m 2 ) completed 2 study visits of 3 identical sessions of fixed‐load (20W) ergometry at fixed pedal rates of 35 or 100 rpm (randomized). Sessions began with 5 min of rest, followed by 3 min of exercise without SubRCO, and immediately transitioning to a 5 min period of exercise+SubRCO at 90 mm Hg bilateral‐thigh cuff inflation pressure. A metabolic system integrated with gas mass‐spectrometry was used to measure breath‐by‐breath VO 2 . Data were linearly interpolated to 1 sec intervals and ensemble averaged across 3 sessions/day for each participant prior to modeling. Models included: single exponential, VO 2REST + A [1 − e −(t‐TD)/τ ]; two exponential, VO 2REST + A [1 − e −(t‐TD)/τ ] + A 2 [1 − e −(t‐TD2)/τ2 ]; O 2 def, [A • 8 min] − ∫ VO 2 dt; MRT, τ + TD; where τ = time constant; A = asymptote; TD = time delay; ∫ VO 2 dt = integral of actual VO 2 across 8 min. Results Single vs two exponential model sums of squares were similar within sessions of 35 (0.05 ± 0.03 vs 0.05 ± 0.03, P = 0.15) and 100 rpm (0.15 ± 0.07 vs 0.14 ± 0.07, P = 0.59), respectively; suggesting an absent Phase III response. Thus, MRT and O 2 def were derived using single exponential models beginning at t = 0. At 35 rpm, MRT = 56 ± 22 sec, and O 2 def = 0.36 ± 0.13 L; which differed significantly from 100 rpm, MRT = 43 ± 13 sec, and O 2 def = 0.70 ± 0.21 L (all P<0.05). Conclusions While these observations demonstrate shortened MRT but increased O 2 def in high versus low pedaling, these data do not suggest locomotor SubRCO provokes worsening of these parameters relating to marked dissociation between VO 2 and muscle O 2 utilization rates during low intensity ergometry in healthy adults. Similarly, although O 2 def demonstrates sensitivity to pedal rate, an absent Phase III rise in VO 2 commensurate with a shortened MRT during high rate pedaling does not suggest an uncoupling of pulmonary and muscle O 2 increases from early to end‐exercise in healthy adults. Support or Funding Information NHLBI RO1 HL126638; AHA 16POST30260021

The electrogenic Na + /HCO 3 − cotransporter (NBCe1/SLC4A4) maintains systemic acid/base balance by reabsorbing HCO 3 − in the renal proximal tubule but also contributes to intracellular pH (pH i ) regulation in the eye, nervous system, pancreas, and intestine. Human recessive mutations lead to diverse phenotypes (glaucoma, proximal renal tubular acidosis, poor dentition) and mouse models suggest some organ manifestations are partially penetrant. The objective of this study was to develop a full spectrum genetically‐encoded live‐imaging toolkit which could be adapted to assay NBCe1‐mediated pH i regulation across a variety of cell types to further characterize how NBCe1 recessive mutations and polymorphisms functionally interact with each other and wild‐type (WT) NBCe1 to produce alterations in cellular acid/base physiology. WT human NBCe1 (WT‐NBCe1) and a recently characterized human recessive mutation (Mut‐NBCe1) were tagged with GFP. Each construct was co‐expressed via transfection in human immortalized trabecular meshwork cells (TM5) with “pHire”, a red‐shifted (556/630nm ex/em) pH‐sensitive variant of mApple, to assay NBCe1 function through real‐time quantification of pH i via widefield epifluorescent live‐imaging. pHire successfully reported pH i of TM5 cells in response to H + loading with NH 4 Cl pulses. Calibration of pHire revealed an in vitro p K a of 7.44, a 5‐fold linear fluorescence response from pH 6.5 to 8.0, and stability following acid loads (pH 5.0) and long imaging protocols (30 min). pH i of TM5 cells fell in response to CO 2 /HCO 3 − — buffered solution and displayed a modest Na + ‐dependent recovery ( dpH i / dt = 0.02 ± 0.01 pH units min −1 in CO 2 /HCO 3 − , −0.06 ± 0.02 in Na + ‐free), consistent with intrinsic TM5‐cell NBCe1 activity. Transfection of WT‐NBCe1 did not consistently alter HCO 3 − recovery but accelerated pH i decrease in Na + ‐free solution by 10‐fold (1017 ± 3%) relative to neighboring untransfected cells. Transfection with E91R‐NBCe1 failed to similarly potentiate acidification upon Na + withdrawal (117 ± 81%), and transfection with Mut‐NBCe1 suppressed Na + ‐dependent acidification to 72 ± 28% of untransfected cells. These data suggest that although both Mut‐NBCe1 and E91R‐NBCe1 are functionally “transporter‐null” they possess different capacities to impair endogenous WT‐NBCe1, possibly through dimerization and localization deficits. Such differences could manifest discretely in specific cell types and may underlie the partial penetrance of some NBCe1‐associated phenotypes. Support or Funding Information Funding: T32‐DK007013 to AJR, DK092408, DK100227 and R25‐DK101405 to MFR.