Sodium/(calcium + potassium) exchanger NCKX4 optimizes KLK4 activity in the enamel matrix microenvironment to regulate ECM modeling.

Enamel development is a process in which extracellular matrix models from a soft proteinaceous matrix to the most mineralized tissue in vertebrates. Patients with mutant NCKX4, a gene encoding a K+-dependent Na+/Ca2+-exchanger, develop a hypomineralized and hypomature enamel. How NCKX4 regulates enamel protein removal to achieve an almost protein-free enamel is unknown. We characterized the upregulation pattern of Nckx4 in the progressively differentiating enamel-forming ameloblasts by qPCR, and as well as confirmed NCKX4 protein to primarily localize at the apical surface of wild-type ruffle-ended maturation ameloblasts by immunostaining of the continuously growing mouse incisors, posing the entire developmental trajectory of enamel. In contrast to the normal mature enamel, where ECM proteins are hydrolyzed and removed, we found significant protein retention in the maturation stage of Nckx4 -/- mouse enamel. The Nckx4 -/- enamel held less Ca2+ and K+ but more Na+ than the Nckx4 +/+ enamel did, as measured by EDX. The alternating acidic and neutral pH zones at the surface of mineralizing Nckx4 +/+ enamel were replaced by a largely neutral pH matrix in the Nckx4 -/- enamel. In situ zymography revealed a reduced kallikrein-related peptidase 4 (KLK4) activity in the Nckx4 -/- enamel. We showed that KLK4 took on 90% of proteinase activity in the maturation stage of normal enamel, and that recombinant KLK4 as well as native mouse enamel KLK4 both performed less effectively in a buffer with increased [Na+] and pH, conditions found in the Nckx4 -/- developing enamel. This study, for the first time to our knowledge, provides evidence demonstrating the impaired in situ KLK4 activity in Nckx4 -/- enamel and suggests a novel function of NCKX4 in facilitating KLK4-mediated hydrolysis and removal of ECM proteins, warranting the completion of enamel matrix modeling.

A sugarcane mosaic virus vector for rapid in planta screening of proteins that inhibit the growth of insect herbivores.

Spodoptera frugiperda (fall armyworm) is a notorious pest that threatens maize production worldwide. Current control measures involve the use of chemical insecticides and transgenic maize expressing Bacillus thuringiensis (Bt) toxins. Although additional transgenes have confirmed insecticidal activity, limited research has been conducted in maize, at least partially due to the technical difficulty of maize transformation. Here, we describe implementation of a sugarcane mosaic virus (SCMV) vector for rapidly testing the efficacy of both endogenous maize genes and heterologous genes from other organisms for the control of S. frugiperda in maize. Four categories of proteins were tested using the SCMV vector: (i) maize defence signalling proteins: peptide elicitors (Pep1 and Pep3) and jasmonate acid conjugating enzymes (JAR1a and JAR1b); (ii) maize defensive proteins: the previously identified ribosome-inactivating protein (RIP2) and maize proteinase inhibitor (MPI), and two proteins with predicted but unconfirmed anti-insect activities, an antimicrobial peptide (AMP) and a lectin (JAC1); (iii) lectins from other plant species: Allium cepa agglutinin (ACA) and Galanthus nivalis agglutinin (GNA); and (iv) scorpion and spider toxins: peptides from Urodacus yaschenkoi (UyCT3 and UyCT5) and Hadronyche versuta (Hvt). In most cases, S. frugiperda larval growth was reduced by transient SCMV-mediated overexpression of genes encoding these proteins. Additionally, experiments with a subset of the SCMV-expressed genes showed effectiveness against two aphid species, Rhopalosiphum maidis (corn leaf aphid) and Myzus persicae (green peach aphid). Together, these results demonstrate that SCMV vectors are a rapid screening method for testing the efficacy and insecticidal activity of candidate genes in maize.

The role of point-of-care ultrasound in the diagnosis of pericardial effusion: a single academic center retrospective study.

BACKGROUND: Symptomatic pericardial effusion (PCE) presents with non-specific features and are often missed on the initial physical exam, chest X-ray (CXR), and electrocardiogram (ECG). In extreme cases, misdiagnosis can evolve into decompensated cardiac tamponade, a life-threatening obstructive shock. The purpose of this study is to evaluate the impact of point-of-care ultrasound (POCUS) on the diagnosis and therapeutic intervention of clinically significant PCE. METHODS: In a retrospective chart review, we looked at all patients between 2002 and 2018 at a major Canadian academic hospital who had a pericardiocentesis for clinically significant PCE. We extracted the rate of presenting complaints, physical exam findings, X-ray findings, ECG findings, time-to-diagnosis, and time-to-pericardiocentesis and how these were impacted by POCUS. RESULTS: The most common presenting symptom was dyspnea (64%) and the average systolic blood pressure (SBP) was 120 mmHg. 86% of people presenting had an effusion > 1 cm, and 89% were circumferential on departmental echocardiogram (ECHO) with 64% having evidence of right atrial systolic collapse and 58% with early diastolic right ventricular collapse. The average time-to-diagnosis with POCUS was 5.9 h compared to > 12 h with other imaging including departmental ECHO. Those who had the PCE identified by POCUS had an average time-to-pericardiocentesis of 28.1 h compared to > 48 h with other diagnostic modalities. CONCLUSION: POCUS expedites the diagnosis of symptomatic PCE given its non-specific clinical findings which, in turn, may accelerate the time-to-intervention.

Pathogens manipulate host autophagy through injected effector proteins.

Macroautophagy/autophagy plays a dual role in many physiological processes of multicellular eukaryotes. In plants, autophagy can be used by both host and pathogen for a beneficiary infection outcome. Plants employ a two-tier innate immune system to defend against invading pathogens. Cell surface localized pattern recognition receptors recognize conserved pathogen-associated molecular patterns (PAMPs) and launch pattern-triggered immunity (PTI) to provide broad-spectrum resistance. Pathogens inject a battery of effector proteins into their hosts to counter PTI and compromise the primary immune response. Hosts induce a second layer of defense called effector-triggered immunity (ETI) to counter the effects of these effectors. In addition to ETI and PTI, autophagy is emerging as a central cellular process modulated by both host and pathogens toward their respective advantage. Pathogens lacking the ability to inject effectors are compromised in virulence. However, molecular targets and biochemical characterization of most of these effector proteins remain elusive. In a recent paper we presented a systematic analysis of interaction between autophagy proteins of Arabidopsis thaliana with effectors from bacterial, fungal, oomycete and nematode pathogens. Abbreviations: ATG, autophagy related; BiFC, bimolecular fluorescence complementation; ETI, effector-triggered immunity; PAMPs, pathogen-associated molecular patterns; PTI, pattern-triggered immunity.

Correction to: Internal Medicine Point of Care Ultrasound: Indicators It's Here to Stay.

This editorial, "Internal Medicine Point of Care Ultrasound: Indicators It's Here to Stay" (DOI: 10.1007/s11606-019-05268-0), was intended to accompany "Education Indicators for Internal Medicine Point-of-Care Ultrasound: a Consensus Report from the Canadian Internal Medicine Ultrasound (CIMUS) Group".

SATB1 establishes ameloblast cell polarity and regulates directional amelogenin secretion for enamel formation.

BACKGROUND: Polarity is necessary for epithelial cells to perform distinct functions at their apical and basal surfaces. Oral epithelial cell-derived ameloblasts at secretory stage (SABs) synthesize large amounts of enamel matrix proteins (EMPs), largely amelogenins. EMPs are unidirectionally secreted into the enamel space through their apical cytoplasmic protrusions, or Tomes' processes (TPs), to guide the enamel formation. Little is known about the transcriptional regulation underlying the establishment of cell polarity and unidirectional secretion of SABs. RESULTS: The higher-order chromatin architecture of eukaryotic genome plays important roles in cell- and stage-specific transcriptional programming. A genome organizer, special AT-rich sequence-binding protein 1 (SATB1), was discovered to be significantly upregulated in ameloblasts compared to oral epithelial cells using a whole-transcript microarray analysis. The Satb1-/- mice possessed deformed ameloblasts and a thin layer of hypomineralized and non-prismatic enamel. Remarkably, Satb1-/- ameloblasts at the secretory stage lost many morphological characteristics found at the apical surface of wild-type (wt) SABs, including the loss of Tomes' processes, defective inter-ameloblastic adhesion, and filamentous actin architecture. As expected, the secretory function of Satb1-/- SABs was compromised as amelogenins were largely retained in cells. We found the expression of epidermal growth factor receptor pathway substrate 8 (Eps8), a known regulator for actin filament assembly and small intestinal epithelial cytoplasmic protrusion formation, to be SATB1 dependent. In contrast to wt SABs, EPS8 could not be detected at the apical surface of Satb1-/- SABs. Eps8 expression was greatly reduced in small intestinal epithelial cells in Satb1-/- mice as well, displaying defective intestinal microvilli. CONCLUSIONS: Our data show that SATB1 is essential for establishing secretory ameloblast cell polarity and for EMP secretion. In line with the deformed apical architecture, amelogenin transport to the apical secretory front and secretion into enamel space were impeded in Satb1-/- SABs resulting in a massive cytoplasmic accumulation of amelogenins and a thin layer of hypomineralized enamel. Our studies strongly suggest that SATB1-dependent Eps8 expression plays a critical role in cytoplasmic protrusion formation in both SABs and in small intestines. This study demonstrates the role of SATB1 in the regulation of amelogenesis and the potential application of SATB1 in ameloblast/enamel regeneration.

Hepatic and portal vein Dopplers in the clinical management of patients with right-sided heart failure: two case reports.

BACKGROUND: Patients with right heart failure pose significant volume management challenges for hemodynamic optimization. We present two cases in which point of care ultrasound (POCUS) of the hepatic and portal veins contributed to the venous hypertension assessment and decongestive strategy for patients with right-sided heart failure. CASE PRESENTATION: Patient A was 91 years old with known pulmonary hypertension and right ventricular systolic dysfunction who presented in septic shock requiring vasopressor support. Hepatic and portal vein Dopplers were consistent with right heart failure and significant venous congestion, therefore, diuresis was initiated which resulted in portal flow normalization, renal recovery, and cessation of vasopressor support. Patient B was 82 years old with severe idiopathic pulmonary fibrosis on home oxygen who presented in decompensated right heart failure. Despite aggressive diuresis, a negative fluid balance was not achieved. The patient continued to deteriorate and prior to their death portal vein, Doppler showed significant flow reversal. CONCLUSION: Hepatic and portal vein Doppler ultrasounds are venous hypertension assessment tools that can be readily used at the bedside by clinicians trained in POCUS that may contribute holistically to the hemodynamic profiling for patients with right heart failure and direct therapeutic interventions.

Sustainability of physical exam skills in a resident-led curriculum in a large internal medicine program with competency based medical education.

BACKGROUND: Competency Based Medical Education (CBME) designates physical examination competency as an Entrustable Professional Activity (EPA). Considerable concern persists regarding the increased time burden CBME may place on educators. We developed a novel physical examination curriculum that shifted the burden of physical examination case preparation and performance assessment from faculty to residents. Our first objective was to determine if participation led to sustainable improvements in physical examination skills. The second objective was to determine if resident peer assessment was comparable to faculty assessment. METHODS: We selected physical exam case topics based on the Objectives of Training in the Specialty of Internal Medicine as prescribed by the Royal College of Physicians and Surgeons of Canada. Internal Medicine residents compiled evidence-based physical exam checklists that faculty reviewed before distribution to all learners. Physical exam practice sessions with whole-group demonstration followed by small-group practice sessions were performed weekly. We evaluated this pilot curriculum with a formative OSCE, during which a resident peer and a faculty member simultaneously observed and assessed examinee performance by. RESULTS: Participation in the novel curriculum practice sessions improved OSCE performance (faculty score mean 78.96 vs. 62.50, p<0.05). Peer assessment overestimated faculty scores (76.2 vs. 65.7, p<0.001), but peer and faculty assessments were highly correlated (R2 = 0.73 (95% CI 0.50-0.87). CONCLUSION: This novel physical examination curriculum leads to sustainable improvement of physical examination skills. Peer assessment correlated well with the gold standard faculty assessment. This resident-led physical examination curriculum enhanced physical examination skills in a CBME environment, with minimal time commitment from faculty members.

Carotid systolic flow time with passive leg raise correlates with fluid status changes in patients undergoing dialysis.

Corrected carotid systolic flow time (CFTc) has been proposed as a measure of volume status in acutely ill patients. This study endeavors to determine whether the change in CFTc with passive leg raise (PLR) maneuver correlates with volume status changes. Dialysis patients at Kingston General Hospital (Kingston, Canada) underwent point-of-care carotid ultrasonography at the beginning and the end of dialysis. With each measurement, 2 values were recorded: the absolute CFTc, and the difference in CFTc before and after the PLR maneuver. A total of 49 measurements were collected during the study period. CFTc changed with PLR by 5±22milliseconds (2.0%) pre-dialysis and by 40±19milliseconds (13.0%) post-dialysis (P<.0001). Incorporating PLR to the CFTc measurement improved the area under the ROC from 0.64 to 0.91. Particularly, in our sample of patients, a 30milliseconds increase in CFTc with PLR predicted the post-dialysis volume state (LR+=11) whereas an increase of less than 20milliseconds argued against it (LR-=0.079). The assessment of CFTc pre- and post-PLR correlates with intravascular volume changes in patients undergoing dialysis. Alternative to the currently available bedside modalities, this technique is non-invasive, objective, simple to perform at the bedside, and reversible with respect to volume challenge.

Simultaneous transcatheter aortic valve replacement and endovascular repair for critical aortic stenosis and large abdominal aortic aneurysm.

A 75-year-old man with severe aortic stenosis, severe chronic obstructive pulmonary disease, NYHA class III heart failure and a large abdominal aortic aneurysm underwent concurrent transfemoral transcatheter aortic valve replacement (TF-TAVR) and endovascular aneurysm repair (EVAR). An Edwards Sapien device was implanted with resolution of hemodynamics. EVAR was performed using an Endurant bifurcated stent graft system. We describe the procedure technique, periprocedural management and one year outcome. To the authors' best knowledge, this is the first case of simultaneous TF-TAVR and EVAR published in North America.