Dual mechanism of brain injury and novel treatment strategy in maple syrup urine disease

Maple syrup urine disease (MSUD) is an inherited disorder of branched-chain amino acid metabolism presenting with lifethreatening cerebral oedema and dysmyelination in affected individuals. Treatment requires life-long dietary restriction and monitoring of branched-chain amino acids to avoid brain injury. Despite careful management, children commonly suffer metabolic decompensation in the context of catabolic stress associated with non-specific illness. The mechanisms underlying this decompensation and brain injury are poorly understood. Using recently developed mouse models of classic and intermediate maple syrup urine disease, we assessed biochemical, behavioural and neuropathological changes that occurred during encephalopathy in these mice. Here, we show that rapid brain leucine accumulation displaces other essential amino acids resulting in neurotransmitter depletion and disruption of normal brain growth and development. A novel approach of administering norleucine to heterozygous mothers of classic maple syrup urine disease pups reduced branched-chain amino acid accumulation in milk as well as blood and brain of these pups to enhance survival. Similarly, norleucine substantially delayed encephalopathy in intermediate maple syrup urine disease mice placed on a high protein diet that mimics the catabolic stress shown to cause encephalopathy in human maple syrup urine disease. Current findings suggest two converging mechanisms of brain injury in maple syrup urine disease including: (i) neurotransmitter deficiencies and growth restriction associated with branchedchain amino acid accumulation and (ii) energy deprivation through Krebs cycle disruption associated with branched-chain ketoacid accumulation. Both classic and intermediate models appear to be useful to study the mechanism of brain injury and potential treatment strategies for maple syrup urine disease. Norleucine should be further tested as a potential treatment to prevent encephalopathy in children with maple syrup urine disease during catabolic stress

Use of Sex-Specific Clinical and Exercise Risk Scores to Identify Patients at Increased Risk for All-Cause Mortality

Importance Risk assessment tools for exercise treadmill testing may have limited external validity. Cardiovascular mortality has decreased in recent decades, and women have been underrepresented in prior cohorts. Objectives To determine whether exercise and clinical variables are associated with differential mortality outcomes in men and women and to assess whether sex-specific risk scores better estimate all-cause mortality. Design, Setting, and Participants This retrospective cohort study included 59 877 patients seen at the Cleveland Clinic Foundation (CCF cohort) from January 1, 2000, through December 31, 2010, and 49 278 patients seen at the Henry Ford Hospital (FIT cohort) from January 1, 1991, through December 31, 2009. All patients were 18 years or older and underwent exercise treadmill testing. Data were analyzed from January 1, 2000, to October 27, 2011, in the CCF cohort and from January 1, 1991, to April 1, 2013, in the FIT cohort. Main Outcomes and Measurements The CCF cohort was divided randomly into derivation and validation samples, and separate risk scores were developed for men and women. Net reclassification, C statistics, and integrated discrimination improvement were used to compare the sex-specific risk scores with other tools that have all-cause mortality as the outcome. Discrimination and calibration were also evaluated with these sex-specific risk scores in the FIT cohort. Results The CCF cohort included 59 877 patients (59.4% men; 40.5% women) with a median (interquartile range [IQR]) age of 54 (45-63) years and 2521 deaths (4.2%) during a median follow-up of 7 (IQR, 4.1-9.6) years. The FIT cohort included 49 278 patients (52.5% men; 47.4% women) with a median (IQR) age of 54 (46-64) years and 6643 deaths (13.5%) during a median (IQR) follow-up of 10.2 (7-13.4) years. C statistics for the sex-specific risk scores in the CCF validation sample were higher (0.79 in women and 0.81 in men) than C statistics using other tools in women (0.70 for Duke Treadmill Score; 0.74 for Lauer nomogram) and men (0.72 for Duke Treadmill Score; 0.75 for Lauer nomogram). Net reclassification and integrated discrimination improvement were superior with the sex-specific risk scores, mostly owing to correct reclassification of events. The sex-specific risk scores in the FIT cohort demonstrated similar discrimination (C statistic, 0.78 for women and 0.79 for men), and calibration was reasonable. Conclusions and Relevance Sex-specific risk scores better estimate mortality in patients undergoing exercise treadmill testing. In particular, these sex-specific risk scores help to identify patients at the highest residual risk in the present era

Double-blind randomized proof-of-concept trial of canakinumab in patients with COVID-19 associated cardiac injury and heightened inflammation

AIMS: In coronavirus disease 2019 (COVID-19), myocardial injury is associated with systemic inflammation and higher mortality. Our aim was to perform a proof of concept trial with canakinumab, a monoclonal antibody to interleukin-1β, in patients with COVID-19, myocardial injury, and heightened inflammation. METHODS AND RESULTS: This trial required hospitalization due to COVID-19, elevated troponin, and a C-reactive protein concentration more than 50 mg/L. The primary endpoint was time to clinical improvement at Day 14, defined as either an improvement of two points on a seven-category ordinal scale or discharge from the hospital. The secondary endpoint was mortality at Day 28. Forty-five patients were randomly assigned to canakinumab 600 mg ( CONCLUSION: There was no difference in time to clinical improvement at Day 14 in patients treated with canakinumab, and no safety concerns were identified. Future studies could focus on high dose canakinumab in the treatment arm and assess efficacy outcomes at Day 28

Conditional Transgenesis Using Dimerizable Cre (DiCre)

Cre recombinase is extensively used to engineer the genome of experimental animals. However, its usefulness is still limited by the lack of an efficient temporal control over its activity. We have recently developed a conceptually new approach to regulate Cre recombinase, that we have called Dimerizable Cre or DiCre. It is based on splitting Cre into two inactive moieties and fusing them to FKBP12 (FK506-binding protein) and FRB (binding domain of the FKBP12-rapamycin associated protein), respectively. These latter can be efficiently hetero-dimerized by rapamycin, leading to the reinstatement of Cre activity. We have been able to show, using in vitro approaches, that this ligand-induced dimerization is an efficient way to regulate Cre activity, and presents a low background activity together with a high efficiency of recombination following dimerization. To test the in vivo performance of this system, we have, in the present work, knocked-in DiCre into the Rosa26 locus of mice. To evaluate the performance of the DiCre system, mice have been mated with indicator mice (Z/EG or R26R) and Cre-induced recombination was examined following activation of DiCre by rapamycin during embryonic development or after birth of progenies. No recombination could be observed in the absence of treatment of the animals, indicating a lack of background activity of DiCre in the absence of rapamycin. Postnatal rapamycin treatment (one to five daily injection, 10 mg/kg i.p) induced recombination in a number of different tissues of progenies such as liver, heart, kidney, muscle, etc. On the other hand, recombination was at a very low level following in utero treatment of DiCre×R26R mice. In conclusion, DiCre has indeed the potentiality to be used to establish conditional Cre-deleter mice. An added advantage of this system is that, contrary to other modulatable Cre systems, it offers the possibility of obtaining regulated recombination in a combinatorial manner, i.e. induce recombination at any desired time-point specifically in cells characterized by the simultaneous expression of two different promoters

Diffusion-Driven Looping Provides a Consistent Framework for Chromatin Organization

Chromatin folding inside the interphase nucleus of eukaryotic cells is done on multiple scales of length and time. Despite recent progress in understanding the folding motifs of chromatin, the higher-order structure still remains elusive. Various experimental studies reveal a tight connection between genome folding and function. Chromosomes fold into a confined subspace of the nucleus and form distinct territories. Chromatin looping seems to play a dominant role both in transcriptional regulation as well as in chromatin organization and has been assumed to be mediated by long-range interactions in many polymer models. However, it remains a crucial question which mechanisms are necessary to make two chromatin regions become co-located, i.e. have them in spatial proximity. We demonstrate that the formation of loops can be accomplished solely on the basis of diffusional motion. The probabilistic nature of temporary contacts mimics the effects of proteins, e.g. transcription factors, in the solvent. We establish testable quantitative predictions by deriving scale-independent measures for comparison to experimental data. In this Dynamic Loop (DL) model, the co-localization probability of distant elements is strongly increased compared to linear non-looping chains. The model correctly describes folding into a confined space as well as the experimentally observed cell-to-cell variation. Most importantly, at biological densities, model chromosomes occupy distinct territories showing less inter-chromosomal contacts than linear chains. Thus, dynamic diffusion-based looping, i.e. gene co-localization, provides a consistent framework for chromatin organization in eukaryotic interphase nuclei

A depauperate immune repertoire precedes evolution of sociality in bees

Background Sociality has many rewards, but can also be dangerous, as high population density and low genetic diversity, common in social insects, is ideal for parasite transmission. Despite this risk, honeybees and other sequenced social insects have far fewer canonical immune genes relative to solitary insects. Social protection from infection, including behavioral responses, may explain this depauperate immune repertoire. Here, based on full genome sequences, we describe the immune repertoire of two ecologically and commercially important bumblebee species that diverged approximately 18 million years ago, the North American Bombus impatiens and European Bombus terrestris. Results We find that the immune systems of these bumblebees, two species of honeybee, and a solitary leafcutting bee, are strikingly similar. Transcriptional assays confirm the expression of many of these genes in an immunological context and more strongly in young queens than males, affirming Bateman’s principle of greater investment in female immunity. We find evidence of positive selection in genes encoding antiviral responses, components of the Toll and JAK/STAT pathways, and serine protease inhibitors in both social and solitary bees. Finally, we detect many genes across pathways that differ in selection between bumblebees and honeybees, or between the social and solitary clades. Conclusions The similarity in immune complement across a gradient of sociality suggests that a reduced immune repertoire predates the evolution of sociality in bees. The differences in selection on immune genes likely reflect divergent pressures exerted by parasites across social contexts