Arabidopsis CML13 and CML14 Have Essential and Overlapping Roles in Plant Development.

Calmodulin (CaM)-like proteins (CMLs) are the largest family of calcium-binding proteins in plants, yet the functions of most CMLs are unknown. Arabidopsis CML13 and CML14 are closely related paralogs that interact with the isoleucine-glutamine (IQ) domains of myosins, IQ-domain proteins and CaM-binding transcription activators (CAMTAs). Here, we explored the physiological roles of CML13 and CML14 during development by using dexamethasone (Dex)-inducible RNA silencing to suppress either CML13 or CML14 transcript levels. In the absence of inducible suppression, CML13- and CML14-RNA-interference lines were indistinguishable from wild-type (WT) plants throughout development. In contrast, induction of silencing treatment led to rapid increases in RNA-hairpin production that correlated with a targeted reduction in CML13 or CML14 transcript levels and a range of developmental and morphological effects. RNA-suppression treatment did not impair the germination of CML13- or 14-RNA-interference lines, but these seedlings were chlorotic, displayed high mortality and failed to achieve seedling establishment. Under Dex treatment, seeds of CML13- and CML14-RNA-interference lines exhibited differential sensitivity to exogenous ABA compared to WT seeds. Induced RNA suppression of mature plants led to reduced silique length, shorter roots and rapid leaf senescence in CML13- and 14-RNA-interference plants, which correlated with increased gene expression of the senescence marker Senescence-Associated Gene13 (SAG13). Plants induced for RNA suppression at 2 weeks post-germination exhibited a much stronger phenotype than treatment of 3-, 4- or 5-week-old plants. Collectively, our data indicate that both CML13 and CML14 are essential for normal development and function across a broad range of tissues and developmental stages.

Arbuscular-Mycorrhizal Symbiosis in Medicago Regulated by the Transcription Factor MtbHLHm1;1 and the Ammonium Facilitator Protein MtAMF1;3.

Root systems of most land plants are colonised by arbuscular mycorrhiza fungi. The symbiosis supports nutrient acquisition strategies predominantly associated with plant access to inorganic phosphate. The nutrient acquisition is enhanced through an extensive network of external fungal hyphae that extends out into the soil, together with the development of fungal structures forming specialised interfaces with root cortical cells. Orthologs of the bHLHm1;1 transcription factor, previously described in soybean nodules (GmbHLHm1) and linked to the ammonium facilitator protein GmAMF1;3, have been identified in Medicago (Medicago truncatula) roots colonised by AM fungi. Expression studies indicate that transcripts of both genes are also present in arbuscular containing root cortical cells and that the MtbHLHm1;1 shows affinity to the promoter of MtAMF1;3. Both genes are induced by AM colonisation. Loss of Mtbhlhm1;1 expression disrupts AM arbuscule abundance and the expression of the ammonium transporter MtAMF1;3. Disruption of Mtamf1;3 expression reduces both AM colonisation and arbuscule development. The respective activities of MtbHLHm1;1 and MtAMF1;3 highlight the conservation of putative ammonium regulators supporting both the rhizobial and AM fungal symbiosis in legumes.

Decreased left heart flow in fetal lambs causes left heart hypoplasia and pro-fibrotic tissue remodeling.

Low blood flow through the fetal left heart is often conjectured as an etiology for hypoplastic left heart syndrome (HLHS). To investigate if a decrease in left heart flow results in growth failure, we generate left ventricular inflow obstruction (LVIO) in mid-gestation fetal lambs by implanting coils in their left atrium using an ultrasound-guided percutaneous technique. Significant LVIO recapitulates important clinical features of HLHS: decreased antegrade aortic valve flow, compensatory retrograde perfusion of the brain and ascending aorta (AAo) from the arterial duct, severe left heart hypoplasia, a non-apex forming LV, and a thickened endocardial layer. The hypoplastic AAo have miRNA-gene pairs annotating to cell proliferation that are inversely differentially expressed by bulk RNA-seq. Single-nucleus RNA-seq of the hypoplastic LV myocardium shows an increase in fibroblasts with a reciprocal decrease in cardiomyocyte nuclei proportions. Fibroblasts, cardiomyocytes and endothelial cells from hypoplastic myocardium have increased expression of extracellular matrix component or fibrosis genes with dysregulated fibroblast growth factor signaling. Hence, a severe sustained ( ~ 1/3 gestation) reduction in fetal left heart flow is sufficient to cause left heart hypoplasia. This is accompanied by changes in cellular composition and gene expression consistent with a pro-fibrotic environment and aberrant induction of mesenchymal programs.

RPG interacts with E3-ligase CERBERUS to mediate rhizobial infection in Lotus japonicus.

Symbiotic interactions between rhizobia and legumes result in the formation of root nodules, which fix nitrogen that can be used for plant growth. Rhizobia usually invade legume roots through a plant-made tunnel-like structure called an infection thread (IT). RPG (Rhizobium-directed polar growth) encodes a coiled-coil protein that has been identified in Medicago truncatula as required for root nodule infection, but the function of RPG remains poorly understood. In this study, we identified and characterized RPG in Lotus japonicus and determined that it is required for IT formation. RPG was induced by Mesorhizobium loti or purified Nodulation factor and displayed an infection-specific expression pattern. Nodule inception (NIN) bound to the RPG promoter and induced its expression. We showed that RPG displayed punctate subcellular localization in L. japonicus root protoplasts and in root hairs infected by M. loti. The N-terminal predicted C2 lipid-binding domain of RPG was not required for this subcellular localization or for function. CERBERUS, a U-box E3 ligase which is also required for rhizobial infection, was found to be localized similarly in puncta. RPG co-localized and directly interacted with CERBERUS in the early endosome (TGN/EE) compartment and near the nuclei in root hairs after rhizobial inoculation. Our study sheds light on an RPG-CERBERUS protein complex that is involved in an exocytotic pathway mediating IT elongation.

Fatal Pediatric Streptococcal Infection: A Clinico-Pathological Study.

PURPOSE AND CONTEXT: Streptococcal Infection (SI) is an important cause of pediatric death in children, yet limited reports exist on autopsy findings in fatal SI cases. METHOD: Case records (1997-2019) of SI with no pre-existing risk factors were reviewed and selected. Their clinical and pathological findings in the autopsy reports were analyzed. RESULTS: In our cohort of 38 cases based on bacterial culture results, SI was most commonly caused by Streptococcus pneumoniae (SPn; 45%) and Streptococcus pyogenes (SPy; 37%). 92% of decedents had some prodromal symptoms prior to terminal presentation. The clinical course was often rapid, with 89% found unresponsive, suddenly collapsing, or dying within 24 hours of hospital admission. 64% of deaths were attributed to sepsis, more frequently diagnosed in the SPy group than in the SPn group (71% vs 48%). Pneumonia was found in both SPn and SPy groups, whereas meningitis was exclusively associated with SPn. CONCLUSION: Our study shows fatal SI is most commonly caused by either SPn or SPy, both of which are frequently associated with prodromal symptoms, rapid terminal clinical course, and evidence of sepsis. Postmortem diagnosis of sepsis is challenging and should be correlated with clinical features, bacterial culture results, and autopsy findings.

Serious and Fatal Complications after Neonatal Circumcision.

Neonatal circumcision (NNC) is the most frequently performed surgical procedure worldwide and is generally considered safe in Western societies. Deaths attributed to NNC are seldom reported and are mostly explained by lack of adherence to medical standards. We reviewed our emergency department database for circumcision-related emergency admissions. During 2000-2013, 19 previously healthy neonates were admitted for acute complications after circumcision. Four were admitted for bleeding, with hemophilia identified in two cases and von Willebrand disease in one. Eight boys required emergency surgery, three for severe bleeding. Four boys with amputation of the glans underwent immediate surgical reconstruction. One infant was taken to the operating room to remove an obstructing Plastibell ring. Seven boys were admitted to the intensive care unit with severe bleeding or sepsis, three of whom ultimately progressed to hemorrhagic or septic shock. Two of these children died of their complications. We estimate that the annual incidence of severe complications requiring hospitalization after NNC in the Greater Toronto Area was approximately 0.01%, and the incidence of fatalities over the 14-yr review period was approximately 0.0012%. Our results indicate that the risk of serious complications and death as a result of NNC is greater than generally assumed.

Paediatric dilated cardiomyopathy with and without endocardial fibroelastosis - a pathological analysis of 89 explants.

Heart failure due to dilated cardiomyopathy is a major indication for paediatric cardiac transplantation. Endocardial fibroelastosis is a recognised pathological finding of unknown prognostic significance in paediatric dilated cardiomyopathy. To evaluate the nature of the association between left ventricular endocardial fibroelastosis and paediatric dilated cardiomyopathy, we reviewed surgical pathology reports of dilated cardiomyopathy explants (1986-2016) in order to characterise the pathological findings and to compare and contrast their frequency among four age groups: less than 1 year; 1-5 years; 6-10 years; and greater than 11 years. The 89 explants (47 males and 42 females) were all characterised by increased weight and left ventricular chamber dilatation without increased wall thickness. Ninety-five per cent of the specimens in the two youngest subsets had left ventricular endocardial fibroelastosis. Compared to the oldest age group, recipients aged 1-5 years had a 6-fold increase and those younger than 1 year a 19-fold increase in the odds of observing left ventricular endocardial fibroelastosis. Explants with and without endocardial fibroelastosis were otherwise phenotypically similar. In paediatric dilated cardiomyopathy endocardial fibroelastosis is a very common pathological finding, especially in infants and young children. We propose that the descriptive, clinico-pathological designation "Dilated Cardiomyopathy with Endocardial Fibroelastosis" should be adopted to facilitate future investigation into the potential prognostic/therapeutic significance of left ventricular endocardial fibroelastosis.

Feasibility of Wearable Haptic Biofeedback Training for Reducing the Knee Abduction Moment During Overground Walking.

Gait modifications are effective in reducing the first peak knee abduction moment (PKAM), a surrogate for knee loading. Reliance on 3D motion capture currently restricts these modifications to the laboratory. Therefore, our purpose was to test the feasibility of a novel wearable biofeedback system to train (1) toe-in and trunk lean modifications and (2) combined toe-in and trunk lean modifications to reduce PKAM during overground walking outside of the laboratory. Twelve healthy participants practiced modifications in a university hallway directly after performing five normal walking trials. The wearable feedback system provided real-time haptic biofeedback during training trials to inform participants if they were within the prescribed modification range (7-12 deg greater than baseline). Participants were instructed to move to the next modification only once they felt comfortable and could perform it with minimal errors. Following training, five trials of each modification were immediately performed in the gait laboratory without feedback. All participants successfully modified their foot progression and trunk angle using the wearable system. At post-test, PKAM decreased from baseline by 62%, 55%, and 28% during combined, trunk leanand toe-in gait, respectively. The wearable feedback system was effective to modify participants' foot and trunk angle by the prescribed amount, resulting in reduced PKAM during all modifications at post-test. Participants were also able to perform a combined modification, although it took longer to report feeling comfortable doing so. This study demonstrates that a wearable feedback system is feasible to modify kinematic parameters and train gait modifications outside the laboratory.

Lossless Compression of Human Movement IMU Signals.

Real-time human movement inertial measurement unit (IMU) signals are central to many emerging medical and technological applications, yet few techniques have been proposed to process and represent this information modality in an efficient manner. In this paper, we explore methods for the lossless compression of human movement IMU data and compute compression ratios as compared with traditional representation formats on a public corpus of human movement IMU signals for walking, running, sitting, standing, and biking human movement activities. Delta coding was the highest performing compression method which compressed walking, running, and biking data by a factor of 10 and compressed sitting and standing data by a factor of 18 relative to the original CSV formats. Furthermore, delta encoding was shown to approach the a posteriori optimal linear compression level. All methods were implemented and released as open source C code using fixed point computation which can be integrated into a variety of computational platforms. These results could serve to inform and enable human movement data compression in a variety of emerging medical and technological applications.

Genetic Diagnosis and the Severity of Cardiovascular Phenotype in Patients With Elastin Arteriopathy.

BACKGROUND: Elastin insufficiency causes recurrent vascular stenoses. Hemizygous deletion of the elastin gene (ELN) causes Williams-Beuren syndrome (WBS), while single nucleotide variants in ELN cause nonsyndromic supravalvar aortic stenosis (SVAS). Our objective was to compare cardiovascular disease outcomes in patients with WBS and nonsyndromic SVAS. METHODS: Patients (81 WBS, 42 nonsyndromic SVAS) with cardiovascular disease were included in this retrospective single center study. Freedom from surgical and catheter interventions and reinterventions was compared. Vascular tissue from 8 patients and 6 controls was analyzed for arterial wall architecture. RESULTS: Patients with nonsyndromic SVAS presented at a younger age (median 0.3 [0.4-0.7] years) compared with patients with WBS (1.3 [0.2-3.0] years) and had lower freedom from surgical/catheter interventions compared with patients with WBS, with median event-free survival 1.1 (0.3-5.9) versus 4.7 (2.4-13.3) years, respectively (hazard ratio, 1.62 [95% CI, 1.02-2.56]; P=0.04). Patients with nonsyndromic SVAS also had a lower freedom from reinterventions (P=0.054 by log-rank test). This was related in part to a higher frequency of primary and reinterventions for concomitant valvar aortic stenosis. Histology revealed abnormal intimal and medial thickening, disorganized and fragmented elastic fibers, reduced smooth muscle calponin expression, and increased macrophage marker, CD68, expression in the arterial walls in patients with WBS and nonsyndromic SVAS compared with controls. CONCLUSIONS: Patients with nonsyndromic SVAS require early and more frequent vascular and valvular interventions and reinterventions, in particular for concomitant valvar aortic stenosis compared with patients with WBS. This provides important prognostic information to guide counseling of affected families with cardiovascular disease and may guide primary intervention strategies based on predicted risk of restenosis.

Mixed-reality view of cardiac specimens: a new approach to understanding complex intracardiac congenital lesions.

Digital reality is an emerging platform for three-dimensional representation of medical imaging data. In this technical innovation paper, the authors evaluated the accuracy and utility of mixed-reality technology in the morphological evaluation of complex congenital heart disease. The authors converted CT datasets of 12 heart specimens with different subtypes of double-outlet right ventricle to stereoscopic images and interrogated them using a mixed-reality system. The morphological features identified on the stereoscopic models were compared with findings at macroscopic examination of the actual heart specimens. The results showed that the mixed-reality system provided highly accurate stereoscopic display of spatially complex congenital cardiac lesions, with interactive features that might enhance 3-D understanding of morphology. Additionally, the authors found that high-resolution digital reproduction of cardiac specimens using clinical CT scanners is feasible for preservation and educational purposes.

Flow-targeted pediatric ex vivo heart perfusion in donation after circulatory death: A porcine model.

BACKGROUND: The optimal blood flow and pressure to perfuse pediatric hearts from donation after circulatory death (DCD) on the ex vivo perfusion system has not been elucidated. This study sought to investigate the optimal perfusion strategy for pediatric DCD hearts by using a juvenile porcine model comparing pressure- vs flow-targeted strategy. METHODS: The hearts of the juvenile DCD pigs were explanted, and the coronary arteries were perfused for 2 hours by the ex vivo heart perfusion system with 2 different perfusion strategies; pressure-targeted perfusion (target coronary perfusion pressure: 40 mm Hg, group A) and flow-targeted perfusion (target coronary perfusion flow: 10 ml/kg/min, group B). The working model heart perfusion was used to assess systolic and diastolic myocardial performance. RESULTS: The body weight, warm and cold ischemic time, and ex vivo perfusion time were comparable between the groups. In the working model, group B showed significantly preserved cardiac output (A: 70.5 ± 15.3 ml/kg/min vs B: 113.8 ± 15.0 ml/kg/min, p < 0.01), stroke volume (A: 0.4 ± 0.1 ml/kg vs B: 0.7 ± 0.1 ml/kg, p < 0.01), and ejection fraction (A: 18.8% ± 5.9% vs B: 35.0% ± 10.6%, p < 0.01). E/e' and Tei index were also significantly preserved in group B. The percentage gain of heart weight after ex vivo (net increase of the heart weight divided by heart weight at baseline) was significantly smaller in group B (A: 20.0% ± 5.3% vs B: 11.6% ± 5.0%, p < 0.05). Troponin-I, myocardial hemorrhage, oxidative stress markers; myeloperoxidase and 8-hydroxy-2'-deoxyguanosine were also significantly lower after ex vivo perfusion in group B (p < 0.05). CONCLUSIONS: The tightly controlled flow-targeted myocardial perfusion strategy for DCD donor hearts achieved better myocardial performance by causing less myocardial edema and limiting myocardial reperfusion injury.

Influence of IMU position and orientation placement errors on ground reaction force estimation.

Wearable inertial measurement units (IMU) have been proposed to estimate GRF outside of specialized laboratories, however the precise influence of sensor placement error on accuracy is unknown. We investigated the influence of IMU position and orientation placement errors on GRF estimation accuracy. METHODS: Kinematic data from twelve healthy subjects based on marker trajectories were used to simulate 1848 combinations of sensor position placement errors (range ±â€¯100 mm) and orientation placement errors (range ±â€¯25°) across eight body segments (trunk, pelvis, left/right thighs, left/right shanks, and left/right feet) during normal walking trials for baseline cases when a single sensor was misplaced and for the extreme cases when all sensors were simultaneously misplaced. Three machine learning algorithms were used to estimate GRF for each placement error condition and compared with the no placement error condition to evaluate performance. RESULTS: Position placement errors for a single misplaced IMU reduced vertical GRF (VGRF), medio-lateral GRF (MLGRF), and anterior-posterior GRF (APGRF) estimation accuracy by up to 1.1%, 2.0%, and 0.9%, respectively and for all eight simultaneously misplaced IMUs by up to 4.9%, 6.0%, and 4.3%, respectively. Orientation placement errors for a single misplaced IMU reduced VGRF, MLGRF, and APGRF estimation accuracy by up to 4.8%, 7.3%, and 1.5%, respectively and for all eight simultaneously misplaced IMUs by up to 20.8%, 23.4%, and 12.3%, respectively. CONCLUSION: IMU sensor misplacement, particularly orientation placement errors, can significantly reduce GRF estimation accuracy and thus measures should be taken to account for placement errors in implementations of GRF estimation via wearable IMUs.

Rebuilding core abscisic acid signaling pathways of Arabidopsis in yeast.

The phytohormone abscisic acid (ABA) regulates plant responses to abiotic stress, such as drought and high osmotic conditions. The multitude of functionally redundant components involved in ABA signaling poses a major challenge for elucidating individual contributions to the response selectivity and sensitivity of the pathway. Here, we reconstructed single ABA signaling pathways in yeast for combinatorial analysis of ABA receptors and coreceptors, downstream-acting SnRK2 protein kinases, and transcription factors. The analysis shows that some ABA receptors stimulate the pathway even in the absence of ABA and that SnRK2s are major determinants of ABA responsiveness by differing in the ligand-dependent control. Five SnRK2s, including SnRK2.4 known to be active under osmotic stress in plants, activated ABA-responsive transcription factors and were regulated by ABA receptor complexes in yeast. In the plant tissue, SnRK2.4 and ABA receptors competed for coreceptor interaction in an ABA-dependent manner consistent with a tight integration of SnRK2.4 into the ABA signaling pathway. The study establishes the suitability of the yeast system for the dissection of core signaling cascades and opens up future avenues of research on ligand-receptor regulation.

A unified multi-kingdom Golden Gate cloning platform.

Assembling composite DNA modules from custom DNA parts has become routine due to recent technological breakthroughs such as Golden Gate modular cloning. Using Golden Gate, one can efficiently assemble custom transcription units and piece units together to generate higher-order assemblies. Although Golden Gate cloning systems have been developed to assemble DNA plasmids required for experimental work in model species, they are not typically applicable to organisms from other kingdoms. Consequently, a typical molecular biology laboratory working across kingdoms must use multiple cloning strategies to assemble DNA constructs for experimental assays. To simplify the DNA assembly process, we developed a multi-kingdom (MK) Golden Gate assembly platform for experimental work in species from the kingdoms Fungi, Eubacteria, Protista, Plantae, and Animalia. Plasmid backbone and part overhangs are consistent across the platform, saving both time and resources in the laboratory. We demonstrate the functionality of the system by performing a variety of experiments across kingdoms including genome editing, fluorescence microscopy, and protein interaction assays. The versatile MK system therefore streamlines the assembly of modular DNA constructs for biological assays across a range of model organisms.

Ankyrin-B dysfunction predisposes to arrhythmogenic cardiomyopathy and is amenable to therapy.

Arrhythmogenic cardiomyopathy (ACM) is an inherited arrhythmia syndrome characterized by severe structural and electrical cardiac phenotypes, including myocardial fibrofatty replacement and sudden cardiac death. Clinical management of ACM is largely palliative, owing to an absence of therapies that target its underlying pathophysiology, which stems partially from our limited insight into the condition. Following identification of deceased ACM probands possessing ANK2 rare variants and evidence of ankyrin-B loss of function on cardiac tissue analysis, an ANK2 mouse model was found to develop dramatic structural abnormalities reflective of human ACM, including biventricular dilation, reduced ejection fraction, cardiac fibrosis, and premature death. Desmosomal structure and function appeared preserved in diseased human and murine specimens in the presence of markedly abnormal ß-catenin expression and patterning, leading to identification of a previously unknown interaction between ankyrin-B and ß-catenin. A pharmacological activator of the WNT/ß-catenin pathway, SB-216763, successfully prevented and partially reversed the murine ACM phenotypes. Our findings introduce what we believe to be a new pathway for ACM, a role of ankyrin-B in cardiac structure and signaling, a molecular link between ankyrin-B and ß-catenin, and evidence for targeted activation of the WNT/ß-catenin pathway as a potential treatment for this disease.

Alveolar capillary dysplasia with misalignment of the pulmonary veins and hypoplastic left heart sequence caused by an in frame deletion within FOXF1.

Alveolar capillary dysplasia with misalignment of the pulmonary veins (ACDMPV) is a rare, autosomal dominant disorder of interstitial lung development, leading to pulmonary hypertension, and death in infancy. Associated features include malformations of the heart, gastrointestinal tract, and genitourinary system. ACDMPV is caused by heterozygous variants in the FOXF1 gene or microdeletions involving FOXF1. We present a male infant with ACDMPV, hypoplastic left heart sequence (HLHS), duodenal atresia, and imperforate anus due to a de novo, in frame deletion in FOXF1: c.209_214del (p.Thr70_Leu71del). Previous reports have suggested that microdeletions involving FOXF1 are associated with ACDMPV with congenital heart defects, including HLHS, gastrointestinal atresias, and other anomalies; whereas likely pathogenic variants within FOXF1 have not been reported with ACDMPV and HLHS. This is the first patient reported with ACDMPV, HLHS, imperforate anus, and duodenal atresia associated with a likely pathogenic variant in the FOXF1 gene.

Influence of skill level on predicting the success of one's own basketball free throws.

Basketball players sometimes claim to know when their shot is good, even before it goes in. This is likely because shooter proprioception can help determine shot outcome, even before their eyes confirm it. This phenomenon, however, has not been systematically explored for collegiate and recreational shooters. This study compared how well collegiate shooters and recreational shooters could predict outcomes of their own free throws without seeing the shot result. Forty collegiate and recreational shooters shot standard free throws while wearing liquid-crystal occlusion glasses that activated to occlude vision immediately following ball release during each shot. After each shot, shooters verbally predicted shot outcome as "in" or "out", and predicted results were compared with actual outcomes. As anticipated, for made shots, collegiate shooters more accurately predicted their own shots than recreational shooters. However, unexpectedly, for missed shots, collegiate shooters were worse than recreational shooters and were even significantly worse than chance. Further analysis found that collegiate shooters exhibited a significantly higher bias toward predicting their shots as "in". Understanding how shooters of different skill levels perceive their own shot could inform future training strategies for improving shooter accuracy.

Investigations After Death in Children.

OBJECTIVES: To discuss the role of investigations after death in children as part of a supplement on "Death and Dying in the PICU." DATA SOURCES: Literature review, personal experience, and expert opinion. DATA SELECTION: Not applicable. DATA EXTRACTION: Moderated by three experts on investigations after death in children. DATA SYNTHESIS: Not relevant. CONCLUSIONS: A multidisciplinary cliniciopathologic conference is important after the death of a child in order to help bring closure to the family and to attempt to address any concerns they may have about the care. It is also an important part of the quality of care process for a tertiary care institution and provides an unique opportunity for ongoing medical education. The model of a multidisciplinary cliniciopathologic conference used by the Ontario Coroner's Office to investigate sudden and unexpected deaths in children under 5 years old, which has been functioning for over 30 years, is described. Reports from this Pediatric Death Review Committee have been influential in improving the care of children in the province of Ontario.