Mitochondria-targeted melatonin photorelease supports the presence of melatonin MT1 receptors in mitochondria inhibiting respiration.

The presence of signaling-competent G protein-coupled receptors in intracellular compartments is increasingly recognized. Recently, the presence of Gi/o protein-coupled melatonin MT1 receptors in mitochondria has been revealed, in addition to the plasma membrane. Melatonin is highly cell permeant, activating plasma membrane and mitochondrial receptors equally. Here, we present MCS-1145, a melatonin derivative bearing a triphenylphosphonium cation for specific mitochondrial targeting and a photocleavable o-nitrobenzyl group releasing melatonin upon illumination. MCS-1145 displayed low affinity for MT1 and MT2 but spontaneously accumulated in mitochondria, where it was resistant to washout. Uncaged MCS-1145 and exogenous melatonin recruited ß-arrestin 2 to MT1 in mitochondria and inhibited oxygen consumption in mitochondria isolated from HEK293 cells only when expressing MT1 and from mouse cerebellum of WT mice but not from MT1-knockout mice. Overall, we developed the first mitochondria-targeted photoactivatable melatonin ligand and demonstrate that melatonin inhibits mitochondrial respiration through mitochondrial MT1 receptors.

Recurrent Cryptogenic Stroke in a Patient With Left Atrial Septal Pouch.

The incomplete fusion of the septum primum and septum secundum results in the formation of the left atrial septal pouch (LASP). The clinical significance of this entity is a matter of controversy; however, it may act as a nidus for thrombus formation. We report a case of a 57-year-old male who was brought to the hospital by his girlfriend due to his bizarre behavior and confusion for one day. The initial workup for his altered mental status did not yield a diagnosis. The patient was admitted for further workup, which included an MRI of the brain that showed numerous very small-sized foci of restricted diffusion involving bilateral cerebral and cerebellar hemispheres consistent with thromboembolic infarct. The patient did not receive a tissue plasminogen activator (TPA) as he was out of the window for TPA. Transthoracic echocardiogram (TTE) with bubble study did not show patent foramen ovale (PFO) or atrial septal defect (ASD). ECG and telemetry showed normal sinus rhythm and no atrial fibrillation. A transesophageal echocardiogram (TEE) was obtained to find the source of the thromboembolic stroke. TEE discovered a 22 x 8-mm cystic structure in the interatrial septum consistent with a LASP. We hypothesize that the LASP may be a risk factor for cryptogenic stroke. Further research is needed to determine the prevalence of atrial septal pouch (ASP) in the general population, its clinical significance, and guidelines for treatment implications.

A Review of the Impella Devices.

The use of mechanical circulatory support (MCS) to provide acute haemodynamic support for cardiogenic shock or to support high-risk percutaneous coronary intervention (HRPCI) has grown over the past decade. There is currently no consensus on best practice regarding its use in these two distinct indications. Impella heart pumps (Abiomed) are intravascular microaxial blood pumps that provide temporary MCS during HRPCI or in the treatment of cardiogenic shock. The authors outline technical specifications of the individual Impella heart pumps and their accompanying technology, the Automated Impella Controller and SmartAssist, their indications for use and patient selection, implantation techniques, device weaning and escalation, closure strategies, anticoagulation regimens, complications, future directions and upcoming trials.

Miniforceps EBUS-guided lymph node biopsy: impact on diagnostic yield.

INTRODUCTION: Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is the standard diagnostic method for sampling mediastinal and hilar lymph nodes. Non-diagnostic samples have led some pulmonologists to add a miniforceps biopsy (EBUS-TBFB) in order to increase diagnostic yield. Our study aims to analyze the impact of adding EBUS-TBFB to the EBUS-TBNA in cases where Rapid On-site Evaluation (ROSE) was negative for malignancy or was non-diagnostic. MATERIAL AND METHODS: This retrospective chart review included 91 patients who were aged 18-90 years old and underwent EBUS with both TBNA and TBFB between January 1, 2013 and July 1, 2018. RESULTS: There was no significant statistical difference in the diagnostic yield of TBNA vs TBFB with a McNemar value of 0.167, and this conclusion was the same when stratified by race, age and lymph node size. Using TBNA as a gold standard, the sensitivity and specificity of TBFB was 87% and 69%, respectively. Out of the non-diagnostic TBNA samples on ROSE and cell-block, subsequent TBFB resulted in additional pathologic diagnoses in 16% of cases, of which 67% were non-caseating granulomas. Furthermore, two additional malignant cases were identified by TBFB consisting of small cell carcinoma and non-Hodgkin's lymphoma. CONCLUSION: In conclusion, TBFB is a useful adjunctive tool in the diagnosis of non-malignant conditions (i.e. granulomatous diseases) with the potential to spare the patient from more invasive surgical biopsies. Training of future fellows in performing TBFB in addition to TBNA should be strongly encouraged.

Association of Operator and Hospital Experience With Procedural Success Rates and Outcomes in Patients Undergoing Percutaneous Coronary Interventions for Chronic Total Occlusions: Insights From the Blue Cross Blue Shield of Michigan Cardiovascular Consortium.

BACKGROUND: An inverse relationship has been described between procedural success and outcomes of all major cardiovascular procedures. However, this relationship has not been studied for percutaneous coronary intervention (PCI) of chronic total occlusion (CTO). METHODS: We analyzed the data on patients enrolled in Blue Cross Blue Shield of Michigan Cardiovascular Consortium registry in Michigan (January 1, 2010 to March 31, 2018) to evaluate the association of operator and hospital experience with procedural success and outcomes of patients undergoing CTO-PCI. CTO-PCI was defined as intervention of a 100% occluded coronary artery presumed to be ≥3 months old. RESULTS: Among 210 172 patients enrolled in the registry, 7389 (3.5%) CTO-PCIs were attempted with a success rate of 53%. CTO-PCI success increased with operator experience (45% and 65% in the lowest and highest experience tertiles) and was the highest for highly experienced operators at higher experience centers and the lowest for inexperienced operators at low experience hospitals. Multivariable logistic regression models (with spline transformed prior operator and institutional experience) demonstrated a positive relationship between prior operator and site experience and procedural success rates (likelihood ratio test=141.12, df=15, P<0.001) but no relationship between operator and site experience and major adverse cardiac event (likelihood ratio test=19.12, df=15, P=0.208). CONCLUSIONS: Operator and hospital CTO-PCI experiences were directly related to procedural success but were not related to major adverse cardiac event among patients undergoing CTO-PCIs. Inexperienced operators at high experience centers had significantly higher success but not major adverse cardiac event rates compared with inexperienced operators at low experience centers. These data suggested that CTO-PCI safety and success could potentially be improved by selective referral of these procedures to experienced operators working at highly experienced centers.

Percutaneous Coronary Intervention for Chronic Total Occlusion-The Michigan Experience: Insights From the BMC2 Registry.

OBJECTIVES: The aim of this study was to describe the performance and outcomes of chronic total occlusion (CTO) percutaneous coronary intervention (PCI) in Michigan. BACKGROUND: CTO PCI has been associated with reduction in angina, but previous registry analyses showed a higher rate of major adverse cardiac events with this procedure. METHODS: To study uptake and outcomes of CTO PCI in Michigan, patients enrolled in the BMC2 (Blue Cross Blue Shield of Michigan Cardiovascular Consortium) registry (2010 to 2017) were evaluated. CTO PCI was defined as intervention in a 100% occluded coronary artery ≥3 months old. RESULTS: Among 210,172 patients enrolled in the registry, 7,389 CTO PCIs (3.5%) were attempted, with 4,614 (58.3%) achieving post-procedural TIMI (Thrombolysis In Myocardial Infarction) flow grade 3. The proportion of PCIs performed on CTOs increased over the study period (from 2.67% in 2010 to 4.48% in 2017). Thirty of 47 hospitals performed >50 CTO interventions in 2017. Pre-procedural angina class ≤2 was present in one-quarter, and functional assessment for ischemia was performed in 46.6% of patients. Major complications occurred in 245 patients (3.3%) and included death (1.4%), post-procedural stroke (0.4%), cardiac tamponade (0.5%), and urgent coronary artery bypass graft surgery (1.3%). Procedural success improved modestly from 44.5% in 2010 to 54.9% in 2017 (p for trend < 0.001). Rates of in-hospital mortality (p for trend = 0.247) and major adverse cardiac event (p for trend = 0.859) for CTO PCI remained unchanged over the study period. CONCLUSIONS: The rate of CTO PCI in Michigan increased over the study period. Although the success rate of CTO PCI has increased modestly in contemporary practice, it remained far below the >80% reported by select high-volume CTO operators. The rate of periprocedural major adverse cardiac events or death remained unchanged over time. These data suggest room for improvement in the selection and functional assessment of CTO lesions before subjecting patients to the increased procedural risk associated with CTO PCI.

Retinoic acid receptor α as a novel contributor to adrenal cortex structure and function through interactions with Wnt and Vegfa signalling.

Primary aldosteronism (PA) is the most frequent form of secondary arterial hypertension. Mutations in different genes increase aldosterone production in PA, but additional mechanisms may contribute to increased cell proliferation and aldosterone producing adenoma (APA) development. We performed transcriptome analysis in APA and identified retinoic acid receptor alpha (RARα) signaling as a central molecular network involved in nodule formation. To understand how RARα modulates adrenal structure and function, we explored the adrenal phenotype of male and female Rarα knockout mice. Inactivation of Rarα in mice led to significant structural disorganization of the adrenal cortex in both sexes, with increased adrenal cortex size in female mice and increased cell proliferation in males. Abnormalities of vessel architecture and extracellular matrix were due to decreased Vegfa expression and modifications in extracellular matrix components. On the molecular level, Rarα inactivation leads to inhibition of non-canonical Wnt signaling, without affecting the canonical Wnt pathway nor PKA signaling. Our study suggests that Rarα contributes to the maintenance of normal adrenal cortex structure and cell proliferation, by modulating Wnt signaling. Dysregulation of this interaction may contribute to abnormal cell proliferation, creating a propitious environment for the emergence of specific driver mutations in PA.

Molecular mechanisms in primary aldosteronism

Primary aldosteronism (PA) is the most common form and an under-diagnosed cause of secondary arterial hypertension, accounting for up to 10% of hypertensive cases and associated to increased cardiovascular risk. PA is caused by autonomous overproduction of aldosterone by the adrenal cortex. It is mainly caused by a unilateral aldosterone-producing adenoma (APA) or bilateral adrenal hyperplasia. Excess aldosterone leads to arterial hypertension with suppressed renin, frequently associated to hypokalemia. Mutations in genes coding for ion channels and ATPases have been identified in APA, explaining the pathophysiology of increased aldosterone production. Different inherited genetic abnormalities led to the distinction of four forms of familial hyperaldosteronism (type I to IV) and other genetic defects very likely remain to be identified. Somatic mutations are identified in APA, but also in aldosterone-producing cell clusters (APCCs) in normal adrenals, in image-negative unilateral hyperplasia, in transitional lesions and in APCC from adrenals with bilateral adrenal hyperplasia (BAH). Whether these structures are precursors of APA or whether somatic mutations occur in a proliferative adrenal cortex, is still a matter of debate. This review will summarize our knowledge on the molecular mechanisms responsible for PA and the recent discovery of new genes related to early-onset and familial forms of the disease. We will also address new issues concerning genomic and proteomic changes in adrenals with APA and discuss adrenal pathophysiology in relation to aldosterone-producing structures in the adrenal cortex.

Molecular genetics of Conn adenomas in the era of exome analysis.

Aldosterone-producing adenomas (APA) are a major cause of primary aldosteronism (PA), the most common form of secondary hypertension. Exome analysis of APA has allowed the identification of recurrent somatic mutations in KCNJ5, CACNA1D, ATP1A1, and ATP2B3 in more than 50 % of sporadic cases. These gain of function mutations in ion channels and pumps lead to increased and autonomous aldosterone production. In addition, somatic CTNNB1 mutations have also been identified in APA. The CTNNB1 mutations were also identified in cortisol-producing adenomas and adrenal cancer, but their role in APA development and the mechanisms specifying the hormonal production or the malignant phenotype remain unknown. The role of the somatic mutations in the regulation of aldosterone production is well understood, while the impact of these mutations on cell proliferation remains to be established. Furthermore, the sequence of events leading to APA formation is currently the focus of many studies. There is evidence for a two-hit model where the somatic mutations are second hits occurring in a previously remodeled adrenal cortex. On the other hand, the APA-driver mutations were also identified in aldosterone-producing cell clusters (APCC) in normal adrenals, suggesting that these structures may represent precursors for APA development. As PA due to APA can be cured by surgical removal of the affected adrenal gland, the identification of the underlying genetic abnormalities by novel biomarkers could improve diagnostic and therapeutic approaches of the disease. In this context, recent data on steroid profiling in peripheral venous samples of APA patients and on new drugs capable of inhibiting mutated potassium channels provide promising preliminary data with potential for translation into clinical care.

Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells.

Immune cells are important components of the tumor microenvironment and influence tumor growth and evolution at all stages of carcinogenesis. Notably, it is now well established that the immune infiltrate in human tumors can correlate with prognosis and response to therapy. The analysis of the immune infiltrate in the tumor microenvironment has become a major challenge for the classification of patients and the response to treatment. The co-expression of inhibitory receptors such as Program Cell Death Protein 1 (PD1; also known as CD279), Cytotoxic T Lymphocyte Associated Protein 4 (CTLA-4), T-Cell Immunoglobulin and Mucin Containing Protein-3 (Tim-3; also known as CD366), and Lymphocyte Activation Gene 3 (Lag-3; also known as CD223), is a hallmark of T cell exhaustion. We developed a multiparametric in situ immunofluorescence staining to identify and quantify at the cellular level the co-expression of these inhibitory receptors. On a retrospective series of frozen tissue of renal cell carcinomas (RCC), using a fluorescence multispectral imaging technology coupled with an image analysis software, it was found that co-expression of PD-1 and Tim-3 on tumor infiltrating CD8+ T cells is correlated with a poor prognosis in RCC. To our knowledge, this represents the first study demonstrating that this automated multiplex in situ technology may have some clinical relevance.

A gain-of-function mutation in the CLCN2 chloride channel gene causes primary aldosteronism.

Primary aldosteronism is the most common and curable form of secondary arterial hypertension. We performed whole-exome sequencing in patients with early-onset primary aldosteronism and identified a de novo heterozygous c.71G>A/p.Gly24Asp mutation in the CLCN2 gene, encoding the voltage-gated ClC-2 chloride channel 1 , in a patient diagnosed at 9 years of age. Patch-clamp analysis of glomerulosa cells of mouse adrenal gland slices showed hyperpolarization-activated Cl- currents that were abolished in Clcn2-/- mice. The p.Gly24Asp variant, located in a well-conserved 'inactivation domain'2,3, abolished the voltage- and time-dependent gating of ClC-2 and strongly increased Cl- conductance at resting potentials. Expression of ClC-2Asp24 in adrenocortical cells increased expression of aldosterone synthase and aldosterone production. Our data indicate that CLCN2 mutations cause primary aldosteronism. They highlight the important role of chloride in aldosterone biosynthesis and identify ClC-2 as the foremost chloride conductor of resting glomerulosa cells.

An Uncommon Variant of an Uncommon Disease: A Caucasian Adolescent with Apical Hypertrophic Cardiomyopathy Diagnosed with Myocardial Perfusion Imaging.

Apical hypertrophic cardiomyopathy (ApHCM) is a subtype of HCM. This variant is more common in the Asian population when compared to North American patients. Patients may present with arrhythmias, heart failure, myocardial infarction, chest discomfort, fatigue, and presyncope or syncope. Initial evaluation requires electrocardiogram and two-dimensional echocardiogram. T-wave inversion in the precordial leads as well as hypertrophy of the left ventricle is hallmarks of the disease. Cardiac magnetic resonance (CMR) imaging is the most specific and sensitive imaging modality. In patients with contraindications for CMR, myocardial perfusion imaging (MPI) has been described to have diagnostic characteristics for ApHCM. MPI images demonstrating a "solar polar" map pattern and increased apical tracer uptake in single-photon emission computed tomography horizontally and vertical long-axis slices are consistent with the diagnosis of ApHCM. Herein, we present a case of a Caucasian adolescent female who underwent a cardiac screening to rule out hypertrophic obstructive cardiomyopathy. Initially, the patient was unable to undergo CMR, and an MPI was utilized to assist with the diagnosis of ApHCM.

Autoimmune hemolytic anemia associated with trimethoprim-sulfamethoxazole use.

PURPOSE: A case report of drug-induced immune hemolytic anemia (DIIHA) triggered by exposure to trimethoprim-sulfamethoxazole is presented along with a brief review of the pathophysiology of DIIHA and diagnostic considerations. SUMMARY: A 58-year-old woman recently initiated on trimethoprim-sulfamethoxazole for treatment of a urinary tract infection presented to the emergency department with generalized weakness and fatigue. Initial laboratory studies were significant for the following values: hemoglobin concentration, 5.6 g/dL (reference range, 12-15 g/dL); mean corpuscular volume, 116.9 µm3 (reference range, 80-100 µm3); and reticulocyte count, 16% (reference range, 0.5-1.5%). An elevated serum lactate dehydrogenase concentration (646 U/L [reference range, 50-150 U/L]) and a low haptoglobin concentration (<10 mg/dL [reference range, 30-200 mg/dL]) indicated a hemolytic process. A peripheral blood smear revealed spherocytosis. Serologic testing showed antibodies to both immunoglobulin G (IgG) and complement component C3b. An antibody identification panel was nonspecifically positive for a warm-reacting autoantibody (IgG). The combination of clinically evident hemolytic anemia, recent exposure to a newly initiated drug, and serologic evidence strongly suggested DIIHA. Trimethoprim-sulfamethoxazole was promptly discontinued, a total of 6 units of packed red blood cells were transfused, and the patient was treated with methylprednisolone sodium succinate. Clinical and hematologic improvements were observed within a few days. Results of follow-up antibody screening and direct antiglobulin testing 4 weeks after discharge were negative. CONCLUSION: A 58-year-old woman developed warm autoimmune hemolytic anemia after receiving trimethoprim-sulfamethoxazole for 5 days.

Thrombotic thrombocytopenic purpura presenting as acute coronary syndrome.

In patients presenting with thrombotic thrombocytopenia purpura and non-ST elevation myocardial infarction, prompt initiation of plasma exchange takes precedence over other invasive diagnostic procedures for coronary artery disease. Such procedures should be delayed until clinical condition and laboratory parameters have been stabilized.

Implications of unexpected diffuse lung uptake on a 99mTc-sestimibi parathyroid scan.

Diffuse (99m)Tc-sestimibi uptake in the lungs is a sign of serious pathology and merits further work-up. We present a case in which diffuse lung uptake was incidentally found on a parathyroid scan.

18F-FDG PET assessment of Lewy body dementia with cerebellar diaschisis.

Alzheimer disease and Lewy body dementia are the 2 most common causes of dementia. Each disease has distinctive regional metabolic reduction patterns on (18)F-FDG PET. In this report, we present a rare case of an elderly man with dementia whereby (18)F-FDG PET clearly showed Lewy body disease with crossed cerebellar diaschisis.

Dramatic expansion and developmental expression diversification of the methuselah gene family during recent Drosophila evolution.

Functional studies of the methuselah/methuselah-like (mth/mthl) gene family have focused on the founding member mth, but little is known regarding the developmental functions of this receptor or any of its paralogs. We undertook a comprehensive analysis of developmental expression and sequence divergence in the mth/mthl gene family. Using in situ hybridization techniques, we detect expression of six genes (mthl1, 5, 9, 11, 13, and 14) in the embryo during gastrulation and development of the gut, heart, and lymph glands. Four receptors (mthl3, 4, 6, and 8) are expressed in the larval central nervous system, imaginal discs, or both, and two receptors (mthl10 and mth) are expressed in both embryos and larvae. Phylogenetic analysis of all mth/mthl genes in five Drosophila species, mosquito and flour beetle structured the mth/mthl family into several subclades. mthl1, 5, and 14 are present in most species, each forming a separate clade. A newly identified Drosophila mthl gene (CG31720; herein mthl15) formed another ancient clade. The remaining Drosophila receptors, including mth, are members of a large "superclade" that diversified relatively recently during dipteran evolution, in many cases within the melanogaster subgroup. Comparing the expression patterns of the mth/mthl "superclade" paralogs to the embryonic expression of the singleton ortholog in Tribolium suggests both subfunctionalization and acquisition of novel functionalities. Taken together, our findings shed novel light on mth as a young member of an adaptively evolving developmental gene family.