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The biofilm formation by bacteria is a complex process that is strongly mediated by various genetic and environmental factors. Biofilms contribute to disease infestation, especially in chronic infections. It is, therefore important to understand the factors affecting biofilm formation. This study reports the role of a functional amyloid curli in biofilm formation at various abiotic surfaces, including medical devices, by an environmental isolate of Enterobacter cloacae (SBP-8). A knockout mutant of csgA, a structural gene of curli, was created to study the effect of curli on biofilm formation by E. cloacae SBP-8. Our findings confirm curli production at 25°C and 37°C in the wild-type strain. We further investigated the role of curli in the attachment of E. cloacae SBP-8 to glass, enteral feeding tube, and foley latex catheter. Contrary to the previous studies reporting the curli production below 30°C in most biofilm-forming bacterial species, we observed its production in E. cloacae SBP-8 at 37°C. The formation of more intense biofilm in wild-type strain on various surfaces compared to curli-deficient strain (ΔcsgA) at both 25°C and 37°C suggested a prominent role of curli in biofilm formation. Further, electron and confocal microscopy studies demonstrated the formation of diffused monolayers of microbial cells on the abiotic surfaces by ΔcsgA strain as compared to the thick biofilm by respective wild-type strain, indicating the involvement of curli in biofilm formation by E. cloacae SBP-8. Overall, our findings provide insight into biofilm formation mediated by curli in E. cloacae SBP-8. Further, we show that it can be expressed at a physiological temperature on all surfaces, thereby indicating the potential role of curli in the pathogenesis.

Parkinson’s Disease (PD) is the second most common neurodegenerative disease affecting 1-2% of the global population with no cure to date. PD is characterized by accumulation of Lewy Bodies (LBs), which are caused due to aggregation of incorrectly folded α-Synuclein, (SNCA). Another form of PD manifestation is characterized by loss of function ofparkin, which encodes an E3 ubiquitin ligase. Despite extensive research, the cause for onset and progression of PD remains unknown and current therapeutics mainly help manage the disease. An alternative line of treatment can be useful.In this study, we have employed two different genetic models ofDrosophilato screen forAyurvediccompounds and found anAyurvedicmercury based organo-metallic drug Ras-Sindoor has neuroprotective function. Our data indicate that characteristic locomotory dysfunction phenotype of PD is restored upon administration of the compound. Interestingly, RS fed flies also exhibit reduced transcript levels of initiator caspasedronc, which possibly prevents cell death in dopaminergic neurons. Additionally, RS fed PD model flies exhibit an enhanced life span. Our studies emphasize beneficial use of traditionalAyurvediccompounds as a holistic cure for PD like multifactorial diseases.

SummaryThe initiator caspase Dronc is the only CARD-domain containing caspase in Drosophila and is essential for apoptosis. Here, we report that homozygous dronc mutant adult animals are short-lived due to the presence of a poorly developed, defective and leaky intestine. Interestingly, this mutant phenotype can be significantly rescued by enteroblast-specific expression of dronc+ in dronc mutant animals, suggesting that proper Drone function specifically in enteroblasts, one of four cell types in the intestine, is critical for normal development of the intestine. Furthermore, enteroblast-specific knockdown of dronc in adult intestines triggers hyperplasia and differentiation defects. These enteroblast-specific functions of Drone do not require the apoptotic pathway and thus occur in a non-apoptotic manner. In summary, we demonstrate that an apoptotic initiator caspase has a very critical non-apoptotic function for normal development and for the control of the cell lineage in the adult midgut and therefore for proper physiology and homeostasis.Highlightsdronc mutants die from a fragile and leaky intestinedronc has a critical function in enteroblasts of the intestinedronc controls proliferation and differentiation in the intestinedronc performs these functions in an apoptosis-independent (non-apoptotic) manner

During organogenesis, cell proliferation is followed by the differentiation of specific cell types to form an organ. Any aberration in differentiation can result in developmental defects, which can result in a partial to a near-complete loss of an organ. We employ the Drosophila eye model to understand the genetic and molecular mechanisms involved in the process of differentiation. In a forward genetic screen, we identified, cullin-4 (cul-4), which encodes an E3 ubiquitin ligase, to play an important role in retinal differentiation. During development, cul-4 is known to be involved in protein degradation, regulation of genomic stability, and regulation of cell cycle. Previously, we have reported that cul-4 regulates cell death during eye development by downregulating Wingless (Wg)/Wnt signaling pathway. We found that loss-of-function of cul-4 results in a reduced eye phenotype, which can be due to onset of cell death. However, we found that loss-of-function of cul-4 also affects retinal development by downregulating retinal determination (RD) gene expression. Early markers of retinal differentiation are dysregulated in cul-4 loss of function conditions, indicating that cul-4 is necessary for differentiation. Furthermore, loss-of-function of cul-4 ectopically induces expression of negative regulators of eye development like Wg and Homothorax (Hth). During eye development, Wg is known to block the progression of a synchronous wave of differentiation referred to as Morphogenetic furrow (MF). In cul-4 loss-of-function background, expression of dpp-lacZ, a MF marker, is significantly downregulated. Our data suggest a new role of cul-4 in retinal differentiation. These studies may have significant bearings on our understanding of early eye development.

A majesty of evolution encompasses in diversity of the types of eyes known today. Out of all organisms, only a bit over 30 different phyla possess sophisticated eyes. Stating that, rest of the animals are not blind. There could be camera type, or compound eyes, or mirror-like eyes, all different from one another in several aspects, still performing more or less the same function. Even though there are several differences, there are striking similarities as well in all diverse animal life forms. After Ramon y Cajal observed striking similarities between vertebrate and insect retinas, over a century ago, newer research keeps adding to the account with the help of cutting edge technology. Today, we know for the fact that there are more similarities than one can think into visual systems of insects and vertebrates. Of all invertebrate model systems, Drosophila melanogaster stands apart; for, it shares a high degree of genetic conservation and less redundancy. Shorter life span, ease of culture, and availability of wide variety of genetic tools are other benefits which make Drosophila melanogaster a great model system. In this chapter, we shall be providing a comparative account on the compound eye of Drosophila melanogaster with that of camera type eye of vertebrates, in terms of development, structure, and physiology.

Genetic mosaic approach is commonly used in the Drosophila eye by completely abolishing or misexpressing a gene within a subset of cells to unravel its role during development. Classical genetic mosaic approach involves random clone generation in all developing fields. Consequently, a large sample size needs to be screened to generate and analyze clones in specific domains of the developing eye. To address domain specific functions of genes during axial patterning, we have developed a system for generating mosaic clones by combining Gal4/UAS and flippase (FLP)/FRT system which will allow generation of loss-of-function as well as gain-of-function clones on the dorsal and ventral eye margins. We used the bifid-Gal4 driver to drive expression of UAS-FLP. This reagent can have multiple applications in (i) studying spatio-temporal function of a gene during dorso-ventral (DV) axis specification in the eye, (ii) analyzing genetic epistasis of genes involved in DV patterning, and (iii) conducting genome wide screens in a domain specific manner.

Patterning in multi-cellular organisms involves progressive restriction of cell fates by generation of boundaries to divide an organ primordium into smaller fields. We have employed the Drosophila eye model to understand the genetic circuitry responsible for defining the boundary between the eye and the head cuticle on the ventral margin. The default state of the early eye is ventral and depends on the function of Lobe (L) and the Notch ligand Serrate (Ser). We identified homothorax (hth) as a strong enhancer of the L mutant phenotype of loss of ventral eye. Hth is a MEIS class gene with a highly conserved Meis-Hth (MH) domain and a homeodomain (HD). Hth is known to bind Extradenticle (Exd) via its MH domain for its nuclear translocation. Loss-of-function of hth, a negative regulator of eye, results in ectopic ventral eye enlargements. This phenotype is complementary to the L mutant phenotype of loss-of-ventral eye. However, if L and hth interact during ventral eye development remains unknown. Here we show that (i) L acts antagonistically to hth, (ii) Hth is upregulated in the L mutant background, and (iii) MH domain of Hth is required for its genetic interaction with L, while its homeodomain is not, (iv) in L mutant background ventral eye suppression function of Hth involves novel MH domain-dependent factor(s), and (v) nuclear localization of Exd is not sufficient to mediate the Hth function in the L mutant background. Further, Exd is not a critical rate-limiting factor for the Hth function. Thus, optimum levels of L and Hth are required to define the boundary between the developing eye and head cuticle on the ventral margin.

During organogenesis in all multi-cellular organisms, axial patterning is required to transform a single layer organ primordium into a three-dimensional organ. The Drosophila eye model serves as an excellent model to study axial patterning. Dorso-ventral (DV) axis determination is the first lineage restriction event during axial patterning of the Drosophila eye. The early Drosophila eye primordium has a default ventral fate, and the dorsal eye fate is established by onset of dorsal selector gene pannier (pnr) expression in a group of cells on the dorsal eye margin. The boundary between dorsal and ventral compartments called the equator is the site for Notch (N) activation, which triggers cell proliferation and differentiation. This review will focus on (1) chronology of events during DV axis determination; (2) how early division of eye into dorsal and ventral compartments contributes towards the growth and patterning of the fly retina, and (3) functions of DV patterning genes. Developmental Dynamics 241:69–84, 2012. V C 2011 Wiley Periodicals, Inc.

Summary Apoptosis-induced proliferation (AiP) is a compensatory mechanism to maintain tissue size and morphology following unexpected cell loss during normal development, and may also be a contributing factor to cancer and drug resistance. In apoptotic cells, caspase-initiated signaling cascades lead to the downstream production of mitogenic factors and the proliferation of neighboring surviving cells. In epithelial cells of Drosophila imaginal discs, the Caspase-9 ortholog Dronc drives AiP via activation of Jun N-terminal kinase (JNK); however, the specific mechanisms of JNK activation remain unknown. Here we show that caspase-induced activation of JNK during AiP depends on an inflammatory response. This is mediated by extracellular reactive oxygen species (ROSs) generated by the NADPH oxidase Duox in epithelial disc cells. Extracellular ROSs activate Drosophila macrophages (hemocytes), which in turn trigger JNK activity in epithelial cells by signaling through the tumor necrosis factor (TNF) ortholog Eiger. We propose that in an immortalized ("undead") model of AiP, signaling back and forth between epithelial disc cells and hemocytes by extracellular ROSs and TNF/Eiger drives overgrowth of the disc epithelium. These data illustrate a bidirectional cell-cell communication pathway with implication for tissue repair, regeneration, and cancer.

Background The progressive neurodegenerative disorder Alzheimer’s disease (AD) manifests as loss of cognitive functions, and finally leads to death of the affected individual. AD may result from accumulation of amyloid plaques. These amyloid plaques comprising of amyloid-beta 42 (Aβ42) polypeptides results from the improper cleavage of amyloid precursor protein (APP) in the brain. The Aβ42 plaques have been shown to disrupt the normal cellular processes and thereby trigger abnormal signaling which results in the death of neurons. However, the molecular-genetic mechanism(s) responsible for Aβ42 mediated neurodegeneration is yet to be fully understood. Methodology/Principal Findings We have utilized Gal4/UAS system to develop a transgenic fruit fly model for Aβ42 mediated neurodegeneration. Targeted misexpression of human Aβ42 in the differentiating photoreceptor neurons of the developing eye of transgenic fly triggers neurodegeneration. This progressive neurodegenerative phenotype resembles Alzheimer’s like neuropathology. We identified a histone acetylase, CREB Binding Protein (CBP), as a genetic modifier of Aβ42 mediated neurodegeneration. Targeted misexpression of CBP along with Aβ42 in the differentiating retina can significantly rescue neurodegeneration. We found that gain-of-function of CBP rescues Aβ42 mediated neurodegeneration by blocking cell death. Misexpression of Aβ42 affects the targeting of axons from retina to the brain but misexpression of full length CBP along with Aβ42 can restore this defect. The CBP protein has multiple domains and is known to interact with many different proteins. Our structure function analysis using truncated constructs lacking one or more domains of CBP protein, in transgenic flies revealed that Bromo, HAT and polyglutamine (BHQ) domains together are required for the neuroprotective function of CBP. This BHQ domain of CBP has not been attributed to promote survival in any other neurodegenerative disorders. Conclusions/Significance We have identified CBP as a genetic modifier of Aβ42 mediated neurodegeneration. Furthermore, we have identified BHQ domain of CBP is responsible for its neuroprotective function. These studies may have significant bearing on our understanding of genetic basis of AD.

All multicellular organisms require axial patterning to transform a single-layer organ primordium to a three-dimensional organ. It involves delineation of anteroposterior (AP), dorsoventral (DV), and proximodistal (PD) axes. Any deviation in this fundamental process results in patterning and growth defects during organogenesis. The Drosophila eye is an excellent model to study axial patterning. In the Drosophila eye, DV lineage is the first axis to be determined, which is followed by generation of the AP axis. The default state of the Drosophila early eye primordium is ventral, and the dorsal fate is established by onset of expression of dorsal eye fate selector pannier (pnr)in a group of cells on the dorsal eye margin. The boundary between dorsal and ventral compartments is the site for activation of Notch (N) signaling and is referred to as the equator. Activation of N signaling is crucial for initiating the cell proliferation and differentiation in the developing Drosophila eye imaginal disc. This chapter will focus on (a) how axial patterning occurs in the developing Drosophila eye, (b) how the developing eye field gets divided into dorsal and ventral cell populations, and (c) how DV patterning genes contribute toward the growth and patterning of the fly retina.

In all multicellular organisms, the fundamental processes of cell proliferation and cell death are crucial for growth regulation during organogenesis. Strict regulation of cell death is important to maintain tissue homeostasis by affecting processes like regulation of cell number, and elimination of unwanted/unfit cells. The developing Drosophila eye is a versatile model to study patterning and growth, where complex signaling pathways regulate growth and cell survival. However, the molecular mechanisms underlying regulation of these processes is not fully understood. In a gain-of-function screen, we found that misexpression of cullin-4 (cul-4), an ubiquitin ligase, can rescue reduced eye mutant phenotypes. Previously, cul-4 has been shown to regulate chromatin remodeling, cell cycle and cell division. Genetic characterization of cul-4 in the developing eye revealed that loss-of-function of cul-4 exhibits a reduced eye phenotype. Analysis of twin-spots showed that in comparison with their wild-type counterparts, the cul-4 loss-of-function clones fail to survive. Here we show that cul-4 clones are eliminated by induction of cell death due to activation of caspases. Aberrant activation of signaling pathways is known to trigger cell death in the developing eye. We found that Wingless (Wg) and c-Jun-amino-terminal-(NH2)-Kinase (JNK) signaling are ectopically induced in cul-4 mutant clones, and these signals co-localize with the dying cells. Modulating levels of Wg and JNK signaling by using agonists and antagonists of these pathways demonstrated that activation of Wg and JNK signaling enhances cul-4 mutant phenotype, whereas downregulation of Wg and JNK signaling rescues the cul-4 mutant phenotypes of reduced eye. Here we present evidences to demonstrate that cul-4 is involved in restricting Wg signaling and downregulation of JNK signaling-mediated cell death during early eye development. Overall, our studies provide insights into a novel role of cul-4 in promoting cell survival in the developing Drosophila eye.

Background Alzheimer's disease (AD) is an age related progressive neurodegenerative disorder. One of the reasons for Alzheimer's neuropathology is the generation of large aggregates of As42 that are toxic in nature and induce oxidative stress, aberrant signaling and many other cellular alterations that trigger neuronal cell death. However, the exact mechanisms leading to cell death are not clearly understood. Methodology/Principal Findings We employed a Drosophila eye model of AD to study how As42 causes cell death. Misexpression of higher levels of As42 in the differentiating photoreceptors of fly retina rapidly induced aberrant cellular phenotypes and cell death. We found that blocking caspase-dependent cell death initially blocked cell death but did not lead to a significant rescue in the adult eye. However, blocking the levels of c-Jun NH (2)-terminal kinase (JNK) signaling pathway significantly rescued the neurodegeneration phenotype of As42 misexpression both in eye imaginal disc as well as the adult eye. Misexpression of As42 induced transcriptional upregulation of puckered (puc), a downstream target and functional read out of JNK signaling. Moreover, a three-fold increase in phospho-Jun (activated Jun) protein levels was seen in As42 retina as compared to the wild-type retina. When we blocked both caspases and JNK signaling simultaneously in the fly retina, the rescue of the neurodegenerative phenotype is comparable to that caused by blocking JNK signaling pathway alone. Conclusions/Significance Our data suggests that (i) accumulation of As42 plaques induces JNK signaling in neurons and (ii) induction of JNK contributes to As42 mediated cell death. Therefore, inappropriate JNK activation may indeed be relevant to the AD neuropathology, thus making JNK a key target for AD therapies.

Axial patterning is crucial for organogenesis. During Drosophila eye development, dorso-ventral (DV) axis determination is the first lineage restriction event. The eye primordium begins with a default ventral fate, on which the dorsal eye fate is established by expression of the GATA-1 transcription factor pannier (pnr). Earlier, it was suggested that loss of pnr function induces enlargement in the dorsal eye due to ectopic equator formation. Interestingly, we found that in addition to regulating DV patterning, pnr suppresses the eye fate by downregulating the core retinal determination genes eyes absent (eya), sine oculis (so) and dacshund (dac) to define the dorsal eye margin. We found that pnr acts downstream of Ey and affects the retinal determination pathway by suppressing eya. Further analysis of the “eye suppression” function of pnr revealed that this function is likely mediated through suppression of the homeotic gene teashirt (tsh) and is independent of homothorax (hth), a negative regulator of eye. Pnr expression is restricted to the peripodial membrane on the dorsal eye margin, which gives rise to head structures around the eye, and pnr is not expressed in the eye disc proper that forms the retina. Thus, pnr has dual function, during early developmental stages pnr is involved in axial patterning whereas later it promotes the head specific fate. These studies will help in understanding the developmental regulation of boundary formation of the eye field on the dorsal eye margin.