Human ERG oncoprotein represses a Drosophila LIM domain binding protein-coding gene Chip.

Human ETS Related Gene, ERG, a master transcription factor, turns oncogenic upon its out-of-context activation in diverse developmental lineages. However, the mechanism underlying its lineage-specific activation of Notch (N), Wnt, or EZH2-three well-characterized oncogenic targets of ERG-remains elusive. We reasoned that deep homology in genetic tool kits might help uncover such elusive cancer mechanisms in Drosophila. By heterologous gain of human ERG in Drosophila, here we reveal Chip, which codes for a transcriptional coactivator, LIM-domain-binding (LDB) protein, as its novel target. ERG represses Drosophila Chip via its direct binding and, indirectly, via E(z)-mediated silencing of its promoter. Downregulation of Chip disrupts LIM-HD complex formed between Chip and Tailup (Tup)-a LIM-HD transcription factor-in the developing notum. A consequent activation of N-driven Wg signaling leads to notum-to-wing transdetermination. These fallouts of ERG gain are arrested upon a simultaneous gain of Chip, sequestration of Wg ligand, and, alternatively, loss of N signaling or E(z) activity. Finally, we show that the human LDB1, a homolog of Drosophila Chip, is repressed in ERG-positive prostate cancer cells. Besides identifying an elusive target of human ERG, our study unravels an underpinning of its lineage-specific carcinogenesis.

Hh signaling from de novo organizers drive lgl neoplasia in Drosophila epithelium.

The Hedgehog (Hh) morphogen regulates growth and patterning. Since Hh signaling is also implicated in carcinogenesis, it is conceivable that de novo Hh-secreting organizers, if formed in association with oncogenic hit could be tumor-cooperative. Here we validate this hypothesis using the Drosophila model of cooperative epithelial carcinogenesis. We generate somatic clones with simultaneous loss of tumor suppressor, Lgl, and gain of the posterior compartment selector, Engrailed (En), known to induce synthesis of Hh. We show that lgl UAS-en clones in the anterior wing compartment trigger Hh signaling cascade via cross-talk with their Ci-expressing wild type cell neighbors. Hh-Dpp signaling from clone boundaries of such ectopically formed de novo organizers in turn drive lgl carcinogenesis. By contrast, Ci-expressing lgl clones transform by autocrine and/or juxtracine activation of Hh signaling in only the posterior compartment. We further show that sequestration of the Hh ligand or loss of Dpp receptor, Tkv, in these Hh-sending or -receiving lgl clones arrested their carcinogenesis. Our results therefore reveal a hitherto unrecognized mechanism of tumor cooperation by developmental organizers, which are induced fortuitously by oncogenic hits.

An efficient method to generate xenograft tumor models of acute myeloid leukemia and hepatocellular carcinoma in adult zebrafish.

Zebrafish is emerging as a promising model for the study of human cancers. Several xenograft models of zebrafish have been developed, particularly in larval stages (<48 h post fertilization) when the immune system of fish is not developed. However, xenografting in adult zebrafish requires laborious and transient methods of immune suppression (γ- irradiation or dexamethasone) that limits engraftment and survival of the tumor or fail to recapitulate specific characteristics of malignancies. Thus, the availability of a simple protocol to successfully engraft adult zebrafish, remains a challenge. The current study addresses this limitation and describes a robust method of xenografting in adult zebrafish. We describe a protocol that involves pre-conditioning of Casper, a pigmentation mutant of zebrafish with busulfan that led to a higher rate of engraftment of hepatocellular carcinoma and acute myeloid leukemia cells. To further ascertain the homing characteristics of the injected cancer cells, we transplanted adult zebrafish by two routes of administration and then studied their compartmentalization. This model presents a valuable alternative to rodents to study the biology of these cancers and also a cost-effective platform for evaluation of potential anti-cancer agents.

NMR Spectroscopy-based Metabolomics of Drosophila Model of Huntington's Disease Suggests Altered Cell Energetics.

Huntington's disease (HD) is a neurodegenerative disorder induced by aggregation of the pathological form of Huntingtin protein that has expanded polyglutamine (polyQ) repeats. In the Drosophila model, for instance, expression of transgenes with polyQ repeats induces HD-like pathologies, progressively correlating with the increasing lengths of these repeats. Previous studies on both animal models and clinical samples have revealed metabolite imbalances during HD progression. To further explore the physiological processes linked to metabolite imbalances during HD, we have investigated the 1D 1H NMR spectroscopy-based metabolomics profile of Drosophila HD model. Using multivariate analysis (PCA and PLS-DA) of metabolites obtained from methanolic extracts of fly heads displaying retinal deformations due to polyQ overexpression, we show that the metabolite imbalance during HD is likely to affect cell energetics. Six out of the 35 metabolites analyzed, namely, nicotinamide adenine dinucleotide (NAD), lactate, pyruvate, succinate, sarcosine, and acetoin, displayed segregation with progressive severity of HD. Specifically, HD progression was seen to be associated with reduction in NAD and increase in lactate-to-pyruvate ratio. Furthermore, comparative analysis of fly HD metabolome with those of mouse HD model and HD human patients revealed comparable metabolite imbalances, suggesting altered cellular energy homeostasis. These findings thus raise the possibility of therapeutic interventions for HD via modulation of cellular energetics.

Epithelial neoplasia in Drosophila entails switch to primitive cell states.

Only select cell types in an organ display neoplasia when targeted oncogenically. How developmental lineage hierarchies of these cells prefigure their neoplastic propensities is not yet well-understood. Here we show that neoplastic Drosophila epithelial cells reverse their developmental commitments and switch to primitive cell states. In a context of alleviated tissue surveillance, for example, loss of Lethal giant larvae (Lgl) tumor suppressor in the wing primordium induced epithelial neoplasia in its Homothorax (Hth)-expressing proximal domain. Transcriptional profile of proximally transformed mosaic wing epithelium and functional tests revealed tumor cooperation by multiple signaling pathways. In contrast, lgl(-) clones in the Vestigial (Vg)-expressing distal wing epithelium were eliminated by cell death. Distal lgl(-) clones, however, could transform when both tissue surveillance and cell death were compromised genetically and, alternatively, when the transcription cofactor of Hippo signaling pathway, Yorkie (Yki), was activated, or when Ras/EGFR signaling was up-regulated. Furthermore, transforming distal lgl(-) clones displayed loss of Vg, suggesting reversal of their terminal cell fate commitment. In contrast, reinforcing a distal (wing) cell fate commitment in lgl(-) clones by gaining Vg arrested their neoplasia and induced cell death. We also show that neoplasia in both distal and proximal lgl(-) clones could progress in the absence of Hth, revealing Hth-independent wing epithelial neoplasia. Likewise, neoplasia in the eye primordium resulted in loss of Elav, a retinal cell marker; these, however, switched to an Hth-dependent primitive cell state. These results suggest a general characteristic of "cells-of-origin" in epithelial cancers, namely their propensity for switch to primitive cell states.

Synthesis of a highly thermostable insulin by phenylalanine conjugation at B29 Lysine.

Globally, millions of diabetic patients require daily life-saving insulin injections. Insulin heat-lability and fibrillation pose significant challenges, especially in parts of the world without ready access to uninterrupted refrigeration. Here, we have synthesized four human insulin analogs by conjugating ε-amine of B29 lysine of insulin with acetic acid, phenylacetic acid, alanine, and phenylalanine residues. Of these, phenylalanine-conjugated insulin, termed FHI, was the most stable under high temperature (65 °C), elevated salt stress (25 mM NaCl), and varying pH levels (ranging from highly acidic pH 1.6 to physiological pH 7.4). It resists fibrillation for a significantly longer duration with sustained biological activity in in vitro, ex vivo, and in vivo and displays prolonged stability over its native counterpart. We further unravel the critical interactions, such as additional aromatic π-π interactions and hydrogen bonding in FHI, that are notably absent in native insulin. These interactions confer enhanced structural stability of FHI and offer a promising solution to the challenges associated with insulin heat sensitivity.

Right ventricular endomyocardial fibrosis.

Endomyocardial fibrosis is a variety of restrictive cardiomyopathy, in which endocardium of one or both ventricles is thickened markedly with involvement of underlying myocardium. Partial obliteration of ventricular cavities by fibrous tissue and thrombus causes diastolic dysfunction with increased resistance to ventricular filling. Systolic function is well preserved till late stages. Biventricular or isolated left ventricular involvement is common. Isolated right ventricular involvement is relatively uncommon. Case reports on endomyocardial fibrosis have declined in literature. In India, endomyocardial fibrosis is mainly reported from Kerala. A case of right ventricular endomyocardial fibrosis from West Bengal is reported here. Isolated right sided endomyocardial fibrosis, massive right atrial enlargement, complete disorganization of tricuspid valve, massive pericardial effusion, normal absolute eosinophil count and its sporadic occurrence outside 15 degrees of the equatorial belt were interesting features in this case of endomyocardial fibrosis. X-ray features were typical of pericardial effusion masking underlying endomyocardial fibrosis. Endomyocardial fibrosis is a neglected research field. It needs more attention from biomedical researchers.

Carotid intima-media thickness in type 2 diabetes mellitus patients with cardiac autonomic neuropathy.

INTRODUCTION: Cardiac autonomic neuropathy (CAN) is an important complication of type 2 diabetes mellitus (T2DM). Accelerated atherosclerosis is also common in T2DM. Carotid intima media thickness (CIMT) is a surrogate marker of atherosclerosis. We conducted a study to assess the CIMT in T2DM patients with CAN. METHODS: In 84 T2DM patients cardiac autonomic function was assessed by five clinical tests including: 1) heart rate variation during deep breathing, 2) hear rate response to standing, 3) Valsalva ratio, 4) postural fall in systolic blood pressure (BP) three minutes after standing, and 5) resting heart rate. CAN was defined as two or more positive tests out of five for cardiac autonomic function. CIMT was measured by two dimensional (2D) ultrasound. We also examined for presence of any atherosclerotic plaque over intima of carotid artery as well as within the carotid bulb. RESULTS: Thirty six (42.85 percent) out of 84 patients were detected to have CAN. CAN was significantly associated with duration of disease after its detection (P = 0.0253), high LDL cholesterol (P = 0.0418), low HDL cholesterol (P = 0.0001), fasting blood sugar (FBS) level (P = 0.0012) and CIMT (P = 0.0001) equal to or more than 69 mm. CONCLUSION: Increased CIMT equal to or more than 69 mm is associated with high occurrence of CAN in diabetic population. Duration of diabetes, abnormal lipid tests and FBS level significantly influence the development of CAN.

Proximate larval epidermal cell layer generates forces for Pupal thorax closure in Drosophila.

During tissue closures, such as embryonic dorsal closure in Drosophila melanogaster, a proximate extra-embryonic layer, amnioserosa, generates forces that drive migration of the flanking lateral embryonic epidermis, thereby zip-shutting the embryo. Arguably, this paradigm of tissue closure is also recapitulated in mammalian wound healing wherein proximate fibroblasts transform into contractile myofibroblasts, develop cell junctions, and form a tissue layer de novo: contraction of the latter then aids in wound closure. Given this parallelism between disparate exemplars, we posit a general principle of tissue closure via proximate cell layer-generated forces. Here, we have tested this hypothesis in pupal thorax closure wherein 2 halves of the presumptive adult thorax of Drosophila, the contralateral heminotal epithelia, migrate over an underlying larval epidermal cell layer. We show that the proximate larval epidermal cell layer promotes thorax closure by its active contraction, orchestrated by its elaborate actomyosin network-driven epithelial cell dynamics, cell delamination, and death-the latter being prefigured by the activation of caspases. Larval epidermal cell dynamics generate contraction forces, which when relayed to the flanking heminota-via their mutual integrin-based adhesions-mediate thorax closure. Compromising any of these contraction force-generating mechanisms in the larval epidermal cell layer slows down heminotal migration, while loss of its relay to the flanking heminota abrogates the thorax closure altogether. Mathematical modeling further reconciles the biophysical underpinning of this emergent mechanism of thorax closure. Revealing mechanism of thorax closure apart, these findings show conservation of an essential principle of a proximate cell layer-driven tissue closure.

Nephropathy in association with annular psoriasis.

Occurrence of glomerular diseases in psoriasis is rare, although the number of reports is increasing in recent years. Different types of glomerular involvement have been reported but mesangioproliferative glomerulonephritis with IgA deposits, AA amyloidosis and membranous nephropathy are relatively common in association with psoriasis. The term 'psoriatic nephropathy' has been introduced recently. We contribute a case to the ongoing discussin regarding psoriatic nephropathy. Our patient had mesangio-proliferative glomerulonephritis (with IgG and C3 deposition) in association with annular psoriasis (rare variety of chronic plaque psoriasis). Presence of messangio-proliferative glomerulonephritis with IgG deposition is rare in association with psoriasis. The patient responded well to weekly methotrexate (15 mg) injection. Methotrexate has not been tried previously in psoriatic nephropathy or reported to be effective in it.

Hypokalemic periodic paralysis due to proximal renal tubular acidosis in a case with membranoproliferative glomerulonephritis.

Proximal renal tubular acidosis (pRTA) is a rare disorder. Hypokalemia may be associated with it; occasionally leading to features like hypokalemic periodic paralysis. Though pRTA is a tubulointerstitial kidney disease, glomerulonephritis may occasionally lead to pRTA by tubular damage through leaking proteins, cytokines or by inflammatory infiltrates. In our reported case a 27 year old male had recurrent episodes of hypokalemic quadriparesis. Investigations revealed features of pRTA including hypokalemia and non-anion-gap hyperchloremic metabolic acidosis. His urine pH dropped to 5 with NH4Cl loading test. Kidney biopsy showed membranoproliferative glomerulonephritis with tubulointerstitial damage. Hypokalemic periodic paralysis and pRTA are uncommon associations of membranoproliferative glomerulonephritis.

Developmental expression patterns of toolkit genes in male accessory gland of Drosophila parallels those of mammalian prostate.

Conservation of genetic toolkits in disparate phyla may help reveal commonalities in organ designs transcending their extreme anatomical disparities. A male accessory sexual organ in mammals, the prostate, for instance, is anatomically disparate from its analogous, phylogenetically distant counterpart - the male accessory gland (MAG) - in insects like Drosophila. It has not been ascertained if the anatomically disparate Drosophila MAG shares developmental parallels with those of the mammalian prostate. Here we show that the development of Drosophila mesoderm-derived MAG entails recruitment of similar genetic toolkits of tubular organs like that seen in endoderm-derived mammalian prostate. For instance, like mammalian prostate, Drosophila MAG morphogenesis is marked by recruitment of fibroblast growth factor receptor (FGFR) - a signalling pathway often seen recruited for tubulogenesis - starting early during its adepithelial genesis. A specialisation of the individual domains of the developing MAG tube, on the other hand, is marked by the expression of a posterior Hox gene transcription factor, Abd-B, while Hh-Dpp signalling marks its growth. Drosophila MAG, therefore, reveals the developmental design of a unitary bud-derived tube that appears to have been co-opted for the development of male accessory sexual organs across distant phylogeny and embryonic lineages. This article has an associated First Person interview with the first author of the paper.

Heterophilic cell-cell adhesion of atypical cadherins Fat and Dachsous regulate epithelial cell size dynamics during Drosophila thorax morphogenesis.

Spatiotemporal changes in epithelial cell sizes-or epithelial cell size dynamics (ECD)-during morphogenesis entail interplay between two opposing forces: cell contraction via actomyosin cytoskeleton and cell expansion via cell-cell adhesion. Cell-cell adhesion-based ECD, however, has not yet been clearly demonstrated. For instance, changing levels of homophilic E-cadherin-based cell-cell adhesion induce cell sorting, but not ECD. Here we show that cell-expansive forces of heterophilic cell-cell adhesion regulate ECD: higher cell-cell adhesion results in cell size enlargement. Thus, ECD during morphogenesis in the heminotal epithelia of Drosophila pupae leading to thorax closure corresponds with spatiotemporal gradients of two heterophilic atypical cadherins-Fat (Ft) and Dachsous (Ds)-and the levels of Ft-Ds heterodimers formed concomitantly. Our mathematical modeling and genetic tests validate this mechanism of dynamic heterophilic cell-cell adhesion-based regulation of ECD. Conservation of these atypical cadherins suggests a wider prevalence of heterophilic cell-cell adhesion-based ECD regulation during animal morphogenesis.

A Drosophila model of oral peptide therapeutics for adult intestinal stem cell tumors.

Peptide therapeutics, unlike small-molecule drugs, display crucial advantages of target specificity and the ability to block large interacting interfaces, such as those of transcription factors. The transcription co-factor of the Hippo pathway, YAP/Yorkie (Yki), has been implicated in many cancers, and is dependent on its interaction with the DNA-binding TEAD/Sd proteins via a large Ω-loop. In addition, the mammalian vestigial-like (VGLL) proteins, specifically their TONDU domain, competitively inhibit YAP-TEAD interaction, resulting in arrest of tumor growth. Here, we show that overexpression of the TONDU peptide or its oral uptake leads to suppression of Yki-driven intestinal stem cell tumors in the adult Drosophila midgut. In addition, comparative proteomic analyses of peptide-treated and untreated tumors, together with chromatin immunoprecipitation analysis, reveal that integrin pathway members are part of the Yki-oncogenic network. Collectively, our findings establish Drosophila as a reliable in vivo platform to screen for cancer oral therapeutic peptides and reveal a tumor suppressive role for integrins in Yki-driven tumors.This article has an associated First Person interview with the first author of the paper.

Discovery of Arginine Ethyl Ester as Polyglutamine Aggregation Inhibitor: Conformational Transitioning of Huntingtin N-Terminus Augments Aggregation Suppression.

Huntington's disease (HD) is a genetic disorder caused by a CAG expansion mutation in the huntingtin gene leading to polyglutamine (polyQ) expansion in the N-terminal part of huntingtin (Httex1). Expanded polyQ, through a complex aggregation pathway, forms aggregates in neurons and presents a potential therapeutic target. Here we show Httex1 aggregation suppression by arginine and arginine ethyl ester (AEE) in vitro, as well as in yeast and mammalian cell models of HD, bearing expanded polyQ. These molecules also rescue locomotion dysfunction in HD Drosophila model. Both molecules alter the hydrogen bonding network of polyQ to enhance its aqueous solubility and delay aggregation. AEE shows direct binding with the NT17 part of Httex1 to induce structural changes to impart an enhanced inhibitory effect. This study provides a platform for the development of better arginine based therapeutic molecules against polyQ-rich Httex1 aggregation.

The leucine zipper motif of the Drosophila AF10 homologue can inhibit PRE-mediated repression: implications for leukemogenic activity of human MLL-AF10 fusions.

In a screen for Drosophila genes that interfere with transcriptional repression mediated by the Polycomb group of genes, we identified a dominant mutation affecting the Alhambra (Alh) gene, the fly homologue of the human AF10 gene. AF10 has been identified as a fusion partner of both MLL and CALM in infant leukemias. Both fusion proteins retain the leucine zipper domain of AF10 but not its PHD domain. We show here that, while the full-length ALH protein has no activity on Polycomb group-responsive elements (PREs), overexpression of the isolated ALH leucine zipper domain activates several PREs. Within the ALH full-length protein, the PHD domain inhibits the PRE deregulation mediated by the leucine zipper domain. This deregulation is conserved in the human AF10 leucine zipper domain, which confers the same activity on an oncogenic MLL-AF10 fusion protein expressed in Drosophila melanogaster. These data reveal new properties for the leucine zipper domain and thus might provide new clues to understanding the mechanisms by which AF10 fusion proteins in which the PHD domain is lost might trigger leukemias in humans.

Selector genes display tumor cooperation and inhibition in Drosophila epithelium in a developmental context-dependent manner.

During animal development, selector genes determine identities of body segments and those of individual organs. Selector genes are also misexpressed in cancers, although their contributions to tumor progression per se remain poorly understood. Using a model of cooperative tumorigenesis, we show that gain of selector genes results in tumor cooperation, but in only select developmental domains of the wing, haltere and eye-antennal imaginal discs of Drosophila larva. Thus, the field selector, Eyeless (Ey), and the segment selector, Ultrabithorax (Ubx), readily cooperate to bring about neoplastic transformation of cells displaying somatic loss of the tumor suppressor, Lgl, but in only those developmental domains that express the homeo-box protein, Homothorax (Hth), and/or the Zinc-finger protein, Teashirt (Tsh). In non-Hth/Tsh-expressing domains of these imaginal discs, however, gain of Ey in lgl- somatic clones induces neoplastic transformation in the distal wing disc and haltere, but not in the eye imaginal disc. Likewise, gain of Ubx in lgl- somatic clones induces transformation in the eye imaginal disc but not in its endogenous domain, namely, the haltere imaginal disc. Our results reveal that selector genes could behave as tumor drivers or inhibitors depending on the tissue contexts of their gains.

Mathematical modeling of sub-cellular asymmetry of fat-dachsous heterodimer for generation of planar cell polarity.

Planar Cell Polarity (PCP) is an evolutionarily conserved characteristic of animal tissues marked by coordinated polarization of cells or structures in the plane of a tissue. In insect wing epithelium, for instance, PCP is characterized by en masse orientation of hairs orthogonal to its apical-basal axis and pointing along the proximal-distal axis of the organ. Directional cue for PCP has been proposed to be generated by complex sets of interactions amongst three proteins - Fat (Ft), Dachsous (Ds) and Four-jointed (Fj). Ft and Ds are two atypical cadherins, which are phosphorylated by Fj, a Golgi kinase. Ft and Ds from adjacent cells bind heterophilically via their tandem cadherin repeats, and their binding affinities are regulated by Fj. Further, in the wing epithelium, sub-cellular levels of Ft-Ds heterodimers are seen to be elevated at the distal edges of individual cells, prefiguring their PCP. Mechanisms generating this sub-cellular asymmetry of Ft-Ds heterodimer in proximal and distal edges of cells, however, have not been resolved yet. Using a mathematical modeling approach, here we provide a framework for generation of this sub-cellular asymmetry of Ft-Ds heterodimer. First, we explain how the known interactions within Ft-Ds-Fj system translate into sub-cellular asymmetry of Ft-Ds heterodimer. Second, we show that this asymmetric localization of Ft-Ds heterodimer is lost when tissue-level gradient of Fj is flattened, or when phosphorylation of Ft by Fj is abolished, but not when tissue-level gradient of Ds is flattened or when phosphorylation of Ds is abrogated. Finally, we show that distal enrichment of Ds also amplifies Ft-Ds asymmetry. These observations reveal that gradient of Fj expression, phosphorylation of Ft by Fj and sub-cellular distal accumulation of Ds are three critical elements required for generating sub-cellular asymmetry of Ft-Ds heterodimer. Our model integrates the known experimental data and presents testable predictions for future studies.