Bidirectional Ventricular Tachycardia in a Young Female: A Case of Andersen-Tawil Syndrome.

Bidirectional ventricular tachycardia (VT) is a rare ventricular dysrhythmia with a limited differential diagnosis that includes digitalis toxicity, catecholaminergic polymorphic VT, aconite poisoning, and genetic channelopathy syndromes, specifically, Andersen-Tawil syndrome (ATS). We present a case of a young female with palpitations found to have bidirectional VT on cardiac event monitor and strong family history of cardiac dysrhythmias. Her physical examination findings included minor dysmorphic features of mandibular hypoplasia, hypertelorism, and clinodactyly. The patient was clinically diagnosed with ATS and started on a beta-blocker for control of ectopy. A second Holter review demonstrated markedly decreased burden of ventricular ectopy compared to the initial monitoring. She was referred for genetic testing, which revealed a KCNJ2 mutation. Bidirectional VT is an uncommon ventricular dysrhythmia that has a limited differential diagnosis, one of which is ATS-a rare genetic disorder that results from mutations in the KCNJ2 gene. The condition is frequently associated with developmental, skeletal, and cardiac abnormalities. Although there are no strong recommendations that exist for treatment of ventricular dysrhythmias associated with this genetic disorder, we demonstrate a case of clinical improvement in a patient with ATS by using the beta-blocker metoprolol succinate. Furthermore, we propose that ATS patients may not need exercise restrictions as overall ventricular ectopy burden decreased with exercise and there was no prolongation of the QT interval. This patient will continue to follow up in our clinic to reassess symptom burden and for continued monitoring for the development of any new features.

The multi-faceted nature of 15 CFTR exonic variations: Impact on their functional classification and perspectives for therapy.

BACKGROUND: The majority of variants of unknown clinical significance (VUCS) in the CFTR gene are missense variants. While change on the CFTR protein structure or function is often suspected, impact on splicing may be neglected. Such undetected splicing default of variants may complicate the interpretation of genetic analyses and the use of an appropriate pharmacotherapy. METHODS: We selected 15 variants suspected to impact CFTR splicing after in silico predictions on 319 missense variants (214 VUCS), reported in the CFTR-France database. Six specialized laboratories assessed the impact of nucleotide substitutions on splicing (minigenes), mRNA expression levels (quantitative PCR), synthesis and maturation (western blot), cellular localization (immunofluorescence) and channel function (patch clamp) of the CFTR protein. We also studied maturation and function of the truncated protein, consecutive to in-frame aberrant splicing, on additional plasmid constructs. RESULTS: Six of the 15 variants had a major impact on CFTR splicing by in-frame (n = 3) or out-of-frame (n = 3) exon skipping. We reclassified variants into: splicing variants; variants causing a splicing defect and the impairment of CFTR folding and/or function related to the amino acid substitution; deleterious missense variants that impair CFTR folding and/or function; and variants with no consequence on the different processes tested. CONCLUSION: The 15 variants have been reclassified by our comprehensive approach of in vitro experiments that should be used to properly interpret very rare exonic variants of the CFTR gene. Targeted therapies may thus be adapted to the molecular defects regarding the results of laboratory experiments.

Functional Roles of Neuronal Nitric Oxide Synthase in Neurodegenerative Diseases and Mood Disorders.

Nitric oxide synthase (NOS) is well known for its involvement in the regulation of the nervous, cardiovascular, and immune systems. Neuronal NOS (nNOS) is the most characterized NOS among all the isoforms. It accounts for most of the production of nitric oxide (NO) in the nervous system required for synaptic transmission and neuroplasticity. Previous studies have described the localization of nNOS in specific brain regions of interest. There is substantial evidence in the literature suggesting that nNOS signaling has significant involvement in several disease pathologies. However, the association between brain nNOS expression profiles and disease remains largely unknown. In this review, we attempt to delineate the contribution of nNOS signaling in memory and mood disorders in order to achieve a better understanding of nNOS in disease modulation.

Receptor Tyrosine Kinases ror1/2 and ryk Are Co-expressed with Multiple Wnt Signaling Components During Early Development of Sea Urchin Embryos.

AbstractA combination of receptors, co-receptors, and secreted Wnt modulators form protein complexes at the cell surface that activate one or more of the three different Wnt signaling pathways (Wnt/ß-catenin, Wnt/JNK, and Wnt/Ca2+). Two or more of these pathways are often active in the same cellular territories, forming Wnt signaling networks; however, the molecular mechanisms necessary to integrate information from these pathways in these situations are unclear in any in vivo model system. Recent studies have implicated two Wnt binding receptor tyrosine kinases, receptor tyrosine kinase-like orphan receptor (Ror) and related-to-receptor tyrosine kinase (Ryk), in the regulation of canonical and non-canonical Wnt signaling pathways, depending on the context; however, the spatiotemporal expression of these genes in relation to Wnt signaling components has not been well characterized in most deuterostome model systems. Here we use a combination of phylogenetic and spatiotemporal gene expression analyses to characterize Ror and Ryk orthologs in sea urchin embryos. Our phylogenetic analysis indicates that both ror1/2 and ryk originated as single genes from the metazoan ancestor. Expression analyses indicate that ror1/2 and ryk are expressed in the same domains of many Wnt ligands and Frizzled receptors essential for the specification and patterning of germ layers along the early anterior-posterior axis. In addition, both genes are co-expressed with Wnt signaling components in the gut, ventral ectoderm, and anterior neuroectoderm territories later in development. Together, our results indicate that Ror and Ryk have a complex evolutionary history and that their spatiotemporal expression suggests that they could contribute to the complexity of Wnt signaling in early sea urchin embryogenesis.

Intercalating TOP2 Poisons Attenuate Topoisomerase Action at Higher Concentrations.

Topoisomerase II (TOP2) poisons are effective cytotoxic anticancer agents that stabilize the normally transient TOP2-DNA covalent complexes formed during the enzyme reaction cycle. These drugs include etoposide, mitoxantrone, and the anthracyclines doxorubicin and epirubicin. Anthracyclines also exert cell-killing activity via TOP2-independent mechanisms, including DNA adduct formation, redox activity, and lipid peroxidation. Here, we show that anthracyclines and another intercalating TOP2 poison, mitoxantrone, stabilize TOP2-DNA covalent complexes less efficiently than etoposide, and at higher concentrations they suppress the formation of TOP2-DNA covalent complexes, thus behaving as TOP2 poisons at low concentration and inhibitors at high concentration. We used induced pluripotent stem cell (iPSC)-derived human cardiomyocytes as a model to study anthracycline-induced damage in cardiac cells. Using immunofluorescence, our study is the first to demonstrate the presence of topoisomerase IIß (TOP2B) as the only TOP2 isoform in iPSC-derived cardiomyocytes. In these cells, etoposide robustly induced TOP2B covalent complexes, but we could not detect doxorubicin-induced TOP2-DNA complexes, and doxorubicin suppressed etoposide-induced TOP2-DNA complexes. In vitro, etoposide-stabilized DNA cleavage was attenuated by doxorubicin, epirubicin, or mitoxantrone. Clinical use of anthracyclines is associated with cardiotoxicity. The observations in this study have potentially important clinical consequences regarding the effectiveness of anticancer treatment regimens when TOP2-targeting drugs are used in combination. These observations suggest that inhibition of TOP2B activity, rather than DNA damage resulting from TOP2 poisoning, may play a role in doxorubicin cardiotoxicity. SIGNIFICANCE STATEMENT: We show that anthracyclines and mitoxantrone act as topoisomerase II (TOP2) poisons at low concentration but attenuate TOP2 activity at higher concentration, both in cells and in in vitro cleavage experiments. Inhibition of type II topoisomerases suppresses the action of other drugs that poison TOP2. Thus, combinations containing anthracyclines or mitoxantrone and etoposide may reduce the activity of etoposide as a TOP2 poison and thus reduce the efficacy of drug combinations.

Osteopontin mediates necroptosis in lung injury after transplantation of ischaemic renal allografts in rats.

BACKGROUND: Respiratory complications after surgery are associated with morbidity and mortality. Acute lung injury can result from the systemic inflammatory response after acute kidney injury. The mechanisms behind this remote injury are not fully understood. In this study, a renal transplantation model was used to investigate remote lung injury and the underlying molecular mechanisms, especially the role of osteopontin (OPN). METHODS: In vitro, human lung epithelial cell line (A549) and monocyte/macrophage cell line (U937) were challenged with tumour necrosis factor-alpha (TNF-α) in combination with OPN. In vivo, the Fischer rat renal grafts were extracted and stored in 4°C University of Wisconsin preserving solution for up to 16 h, and transplanted into Lewis rat recipients. Lungs were harvested on Day 1 after grafting for further analysis. RESULTS: Renal engraftment was associated with pathological changes and an increase in TNF-α and interleukin-1 beta in the lung of the recipient. OPN, endoplasmic reticulum (ER) stress, and necroptosis were increased in both the recipient lung and A549 cells challenged with TNF-α. Exogenous OPN exacerbated lung injury and necroptosis. Suppression of OPN through siRNA reduced remote lung injury by mitigation of ER stress, necroptosis, and the inflammatory response. CONCLUSIONS: Renal allograft transplant triggers recipient remote lung injury, which is, in part, mediated by OPN signalling. This study may provide a molecular basis for strategies to be developed to treat such perioperative complications.

TOP2B: The First Thirty Years.

Type II DNA topoisomerases (EC 5.99.1.3) are enzymes that catalyse topological changes in DNA in an ATP dependent manner. Strand passage reactions involve passing one double stranded DNA duplex (transported helix) through a transient enzyme-bridged break in another (gated helix). This activity is required for a range of cellular processes including transcription. Vertebrates have two isoforms: topoisomerase IIα and ß. Topoisomerase IIß was first reported in 1987. Here we review the research on DNA topoisomerase IIß over the 30 years since its discovery.

Deciphering the molecular basis of ferroportin resistance to hepcidin: Structure/function analysis of rare SLC40A1 missense mutations found in suspected hemochromatosis type 4 patients.

Genetic medicine applied to the study of hemochromatosis has identified the systemic loop controlling iron homeostasis, centered on hepcidin-ferroportin interaction. Current challenges are to dissect the molecular pathways underlying liver hepcidin synthesis in response to circulatory iron, HFE, TFR2, HJV, TMPRSS6 and BMP6 functions, and to define the major structural elements of hepcidin-ferroportin interaction. We built a first 3D model of human ferroportin structure, using the crystal structure of EmrD, a bacterial drug efflux transporter of the Major Facilitator Superfamily, as template. The model enabled study of disease-associated mutations, and guided mutagenesis experiments to determine the role of conserved residues in protein stability and iron transport. Results revealed novel amino acids that are critical for the iron export function and the hepcidin-mediated inhibition mechanism: for example, tryptophan 42, localized in the extracellular end of the ferroportin pore and involved in both biological functions. Here, we propose a strategy that is not limited to structure analysis, but integrates information from different sources, including human disease-associated mutations and functional in vitro assays. The first major hypothesis of this PhD thesis is that ferroportin resistance to hepcidin relies on different molecular mechanisms that are critical for ferroportin endocytosis, and include at least three fundamental steps: (i) hepcidin binding to ferroportin, (ii) structural reorganization of the N- and C-ter ferroportin lobes, and (iii) ferroportin ubiquitination.

Proteasomal inhibition potentiates drugs targeting DNA topoisomerase II.

The reaction mechanism of DNA topoisomerase II (TOP2) involves a covalent double-strand break intermediate in which the enzyme is coupled to DNA via a 5'-phosphotyrosyl bond. This normally transient enzyme-bridged break is stabilised by drugs such as mitoxantrone, mAMSA, etoposide, doxorubicin, epirubicin and idarubicin, which are referred to as TOP2 poisons. Removal of topoisomerase II by the proteasome is involved in the repair of these lesions. In K562 cells, inhibiting the proteasome with MG132 significantly potentiated the growth inhibition by these six drugs that target topoisomerase II, and the highest level of potentiation was observed with mitoxantrone. Mitoxantrone also showed the greatest potentiation by MG132 in three Nalm 6 cell lines with differing levels of TOP2A or TOP2B. Mitoxantrone was also potentiated by the clinically used proteasome inhibitor PS341 (Velcade). We have also shown that proteasome inhibition with MG132 in K562 cells reduces the rate of removal of mitoxantrone or etoposide stabilised topoisomerase complexes from DNA, suggesting a possible mechanism for the potentiation of topoisomerase II drugs by proteasomal inhibition.

Differential Targeting of Human Topoisomerase II Isoforms with Small Molecules.

The TOP2 poison etoposide has been implicated in the generation of secondary malignancies during cancer treatment. Structural similarities between TOP2 isoforms challenge the rational design of isoform-specific poisons to further delineate these processes. Herein, we describe the synthesis and biological evaluation of a focused library of etoposide analogues, with the identification of two novel small molecules exhibiting TOP2B-dependent toxicity. Our findings pave the way toward studying isoform-specific cellular processes by means of small molecule intervention.

Targeting zero non-attendance in healthcare clinics.

Non-attendance represents a significant cost to many health systems, resulting in inefficiency, wasted resources, poorer service delivery and lengthened waiting queues. Past studies have considered extensively the reasons for non-attendance and have generally concluded that the use of reminder systems is effective. Despite this, there will always be a certain level of non-attendance arising from unforeseeable and unpreventable circumstances, such as illness or accidents, leading to unfilled appointments. This paper reviews current approaches to the non-attendance problem, and presents a high-level approach to fill last minute appointments arising out of unforeseeable non-attendance. However, no single approach will work for all clinics and implementation of these ideas must occur at a local level. These approaches include use of social networks, such as Twitter and Facebook, as a communication tool in order to notify prospective patients when last-minute appointments become available. In addition, teleconsultation using video-conferencing technologies would be suitable for certain last-minute appointments where travel time would otherwise be inhibiting. Developments of new and innovative technologies and the increasing power of social media, means that zero non-attendance is now an achievable target. We hope that this will lead to more evidence-based evaluations from the implementation of these strategies in various settings at a local level.

Design of a large scale community-based self-management system for diabetes mellitus.

This paper presents the ongoing development of a community based, self-management system for diabetes mellitus, which incorporates web-based, SMS and mobile-terminal functionalities. SMS represents the first stage of our system development as it is widely available on all mobile phones, convenient and becoming increasingly popular. We discuss past findings and the need for such a system, as well as design, implementation and system architecture. Poor self-management of diabetes is associated with progression into more complex health issues, manifesting as a significant public health burden and impacting negatively on an individual's quality of life. This approach recognizes that telehealth systems will play an increasingly pertinent role in health systems worldwide.

Durable, region-specific protein patterning in microfluidic channels.

We present a straightforward, accessible method to covalently pattern proteins in poly(dimethyl siloxane) (PDMS) microchannels. Our approach includes (i) region-specific photografting of a layer of poly(acrylamide) (PAAm) and (ii) bioconjugation of PAAm with a desired protein. The method produces symmetric protein patterns on all channel walls, which have high specificity and pattern fidelity, are compatible with a variety of geometries and exhibit excellent longevity under shear stresses of up to 1 dyn/cm. We demonstrate the generality of the method by creating multi-protein gradients within microfluidic microchannels and by in-situ patterning of islands of multiple proteins. Protein activity was observed by the digestion of BODIPY-casein using channels patterned with trypsin.

Genetic polymorphisms and plasma levels of transforming growth factor-beta(1) in Chinese patients with tuberculosis in Hong Kong.

Susceptibility to tuberculosis (TB) may be affected by host genetic factors. Elevated levels of transforming growth factor-beta 1 (TGF-beta(1)) were found in plasma of patients with active TB compared with those of healthy contacts. To investigate the association of TGF-beta(1) gene polymorphisms (C-509T and T869C) and plasma levels with the risk of TB in Hong Kong Chinese adults, a case-control study was carried out on 174 active TB patients and 174 healthy controls matched for age, gender and smoking. Blood samples from 180 blood donors served as another control group. Genotyping was carried out on genomic DNA using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Plasma TGF-beta(1) was measured by commercially available ELISA kit. We found no differences in the distribution of genotypes or alleles of TGF-beta(1) gene polymorphisms at C-509T and T869C between patients and either group of healthy controls. Patients with TB had elevated plasma TGF-beta(1) levels compared with healthy controls irrespective of their genotypes (p<0.001). In conclusion, TGF-beta(1) gene polymorphism at C-509T and T869C is not associated with TB susceptibility in Hong Kong Chinese adults, but elevated plasma TGF-beta(1) levels suggests that this cytokine may play a role in the pathogenesis of tuberculosis.

Digital PCR for the molecular detection of fetal chromosomal aneuploidy.

Trisomy 21 is the most common reason that women opt for prenatal diagnosis. Conventional prenatal diagnostic methods involve the sampling of fetal materials by invasive procedures such as amniocentesis. Screening by ultrasonography and biochemical markers have been used to risk-stratify pregnant women before definitive invasive diagnostic procedures. However, these screening methods generally target epiphenomena, such as nuchal translucency, associated with trisomy 21. It would be ideal if noninvasive genetic methods were available for the direct detection of the core pathology of trisomy 21. Here we outline an approach using digital PCR for the noninvasive detection of fetal trisomy 21 by analysis of fetal nucleic acids in maternal plasma. First, we demonstrate the use of digital PCR to determine the allelic imbalance of a SNP on PLAC4 mRNA, a placenta-expressed transcript on chromosome 21, in the maternal plasma of women bearing trisomy 21 fetuses. We named this the digital RNA SNP strategy. Second, we developed a nonpolymorphism-based method for the noninvasive prenatal detection of trisomy 21. We named this the digital relative chromosome dosage (RCD) method. Digital RCD involves the direct assessment of whether the total copy number of chromosome 21 in a sample containing fetal DNA is overrepresented with respect to a reference chromosome. Even without elaborate instrumentation, digital RCD allows the detection of trisomy 21 in samples containing 25% fetal DNA. We applied the sequential probability ratio test to interpret the digital PCR data. Computer simulation and empirical validation confirmed the high accuracy of the disease classification algorithm.