How antisense transcripts can evolve to encode novel proteins.

Protein coding features can emerge de novo in non coding transcripts, resulting in emergence of new protein coding genes. Studies across many species show that a large fraction of evolutionarily novel non-coding RNAs have an antisense overlap with protein coding genes. The open reading frames (ORFs) in these antisense RNAs could also overlap with existing ORFs.

Modeling Length Changes in De Novo Open Reading Frames during Neutral Evolution.

For protein coding genes to emerge de novo from a non-genic DNA, the DNA sequence must gain an open reading frame (ORF) and the ability to be transcribed. The newborn de novo gene can further evolve to accumulate changes in its sequence. Consequently, it can also elongate or shrink with time.

Neutral Models of De Novo Gene Emergence Suggest that Gene Evolution has a Preferred Trajectory.

New protein coding genes can emerge from genomic regions that previously did not contain any genes, via a process called de novo gene emergence. To synthesize a protein, DNA must be transcribed as well as translated. Both processes need certain DNA sequence features.

Bacterial Hsp90 predominantly buffers but does not potentiate the phenotypic effects of deleterious mutations during fluorescent protein evolution.

Chaperones facilitate the folding of other ("client") proteins and can thus affect the adaptive evolution of these clients. Specifically, chaperones affect the phenotype of proteins via two opposing mechanisms. On the one hand, they can buffer the effects of mutations in proteins and thus help preserve an ancestral, premutation phenotype.

GroEL/S Overexpression Helps to Purge Deleterious Mutations and Reduce Genetic Diversity during Adaptive Protein Evolution.

Chaperones are proteins that help other proteins fold. They also affect the adaptive evolution of their client proteins by buffering the effect of deleterious mutations and increasing the genetic diversity of evolving proteins. We study how the bacterial chaperone GroE (GroEL+GroES) affects the evolution of green fluorescent protein (GFP).

Identification of novel circadian transcripts in the zebrafish retina.

High fecundity, transparent embryos for monitoring the rapid development of organs and the availability of a well-annotated genome has made zebrafish a model organism of choice for developmental biology and neurobiology. This vertebrate model, which is also a favourite in chronobiology studies, shows striking circadian rhythmicity in behaviour. Here, we identify novel genes in the zebrafish genome that are expressed in the zebrafish retina.

Systematic comparison of the response properties of protein and RNA mediated gene regulatory motifs.

We present a framework enabling the dissection of the effects of motif structure (feedback or feedforward), the nature of the controller (RNA or protein), and the regulation mode (transcriptional, post-transcriptional or translational) on the response to a step change in the input. We have used a common model framework for gene expression where both motif structures have an activating input and repressing regulator, with the same set of parameters, to enable a comparison of the responses. We studied the global sensitivity of the system properties, such as steady-state gain, overshoot, peak time, and peak duration, to parameters.

Microfluidic devices for imaging neurological response of Drosophila melanogaster larva to auditory stimulus.

Two microfluidic devices (pneumatic chip and FlexiChip) have been developed for immobilization and live-intact fluorescence functional imaging of Drosophila larva's Central Nervous System (CNS) in response to controlled acoustic stimulation. The pneumatic chip is suited for automated loading/unloading and potentially allows high throughput operation for studies with a large number of larvae while the FlexiChip provides a simple and quick manual option for animal loading and is suited for smaller studies. Both chips were capable of significantly reducing the endogenous CNS movement while still allowing the study of sound-stimulated CNS activities of Drosophila 3rd instar larvae using genetically encoded calcium indicator GCaMP5.

Non-coding RNA interact to regulate neuronal development and function.

The human brain is one of the most complex biological systems, and the cognitive abilities have greatly expanded compared to invertebrates without much expansion in the number of protein coding genes. This suggests that gene regulation plays a very important role in the development and function of nervous system, by acting at multiple levels such as transcription and translation. In this article we discuss the regulatory roles of three classes of non-protein coding RNAs (ncRNAs)-microRNAs (miRNAs), piwi-interacting RNA (piRNAs) and long-non-coding RNA (lncRNA), in the process of neurogenesis and nervous function including control of synaptic plasticity and potential roles in neurodegenerative diseases.

The melanocyte photosensory system in the human skin.

The pigment cells form the largest population of neural crest cells to migrate into the epidermis and hair follicle along each dermatomic area from the neural folds. The melanopsin system responsible for photoentrainment, was isolated from the photosensitive dermal melanophores of frogs Xenopus laevis responding to light. Melanocytes form a photoresponsive network which reads the environmental seasonal variations in the light cycles in the same manner.

Silencing synaptic communication between random interneurons during Drosophila larval locomotion.

Genetic manipulation of individual neurons provides a powerful approach toward understanding their contribution to stereotypic behaviors. We describe and evaluate a method for identifying candidate interneurons and associated neuropile compartments that mediate Drosophila larval locomotion. We created Drosophila larvae that express green fluorescent protein (GFP) and a shibire(ts1) (shi(ts1)) transgene (a temperature-sensitive neuronal silencer) in small numbers of randomly selected cholinergic neurons.

Embryonic vasculogenesis in nodular melanomas and tumour differentiation.

The relationship of vasculogenic mimicry to pigment in nodular vertical growth phase [VGP] cutaneous melanomas is assessed in this study. 10 nodules each from 27 tumors, 15 pigmented and 12 amelanotic were sampled in proportion to the pigment level. Serial frozen and paraffin sections subjected to HE, Reticulin, PAS to assess the vascular pattern; Dopa Oxidase and Immunopositivity for HMB45, LN5 [laminin 5] & integrin[α(5)β(1)], and EM [electron microscopy] to identify Weibel-Palade bodies within endothelial cells.

Patterns of neural differentiation in melanomas.

Background: Melanomas, highly malignant tumors arise from the melanocytes which originate as multipotent neural crest cells during neural tube genesis. The purpose of this study is to assess the pattern of neural differentiation in relation to angiogenesis in VGP melanomas using the tumor as a three dimensional system.

Methods: Tumor-vascular complexes [TVC] are formed at the tumor-stroma interphase, by tumor cells ensheathing angiogenic vessels to proliferate into a mantle of 5 to 6 layers [L1 to L5] forming a perivascular mantle zone [PMZ].

Frequenin/NCS-1 and the Ca2+-channel alpha1-subunit co-regulate synaptic transmission and nerve-terminal growth.

Drosophila Frequenin (Frq) and its mammalian and worm homologue, NCS-1, are Ca(2+)-binding proteins involved in neurotransmission. Using site-specific recombination in Drosophila, we created two deletions that removed the entire frq1 gene and part of the frq2 gene, resulting in no detectable Frq protein. Frq-null mutants were viable, but had defects in larval locomotion, deficient synaptic transmission, impaired Ca(2+) entry and enhanced nerve-terminal growth.

Modular neuropile organization in the Drosophila larval brain facilitates identification and mapping of central neurons.

Elucidating how neuronal networks process information requires identification of critical individual neurons and their connectivity patterns. For this purpose, we used the third-instar Drosophila larval brain and applied reverse-genetic tools, immunolabeling procedures, and 3D digital reconstruction software. Consistent topological definition of neuropile compartments in the larval brain can be obtained through simple fluorescence-immunolabeling methods.

Photic input pathways that mediate the Drosophila larval response to light and circadian rhythmicity are developmentally related but functionally distinct.

The Drosophila melanogaster larval photosensory organ that mediates the response to light consists of bilaterally symmetrical clusters of 12 photoreceptors. These are distinguished on the basis of expression of the rhodopsins Rh5 and Rh6. The Rh6-expressing cells correspond to the Hofbauer-Buchner (H-B) eyelet found later in the posterior margin of the adult compound eye and recently shown to function as an input pathway in the entrainment of circadian rhythmicity in adult Drosophila.

Chemical basis for the biological activity of imexon and related cyanoaziridines.

Chemical aspects of mode of action of imexon and related cyanoaziridines were studied. These compounds do not alkylate DNA nor react with the epsilon-amino groups of l-lysine, despite the presence of an aziridine ring. They do react readily with biologically important sulfhydryl compounds to give products derived from either aziridine ring opening, interaction with the cyano group of cyanoaziridines, or opening of the iminopyrrolidone ring of imexon.

The accessory subunit of DNA polymerase gamma is essential for mitochondrial DNA maintenance and development in Drosophila melanogaster.

DNA polymerase gamma, Pol gamma, is the key replicative enzyme in animal mitochondria. The Drosophila enzyme is a heterodimer comprising catalytic and accessory subunits of 125 kDa and 35 kDa, respectively. Both subunits have been cloned and characterized in a variety of model systems, and genetic mutants of the catalytic subunit were first identified in Drosophila, as chemically induced mutations that disrupt larval behavior (tamas).

Drosophila Amphiphysin is a post-synaptic protein required for normal locomotion but not endocytosis.

Clathrin-mediated endocytosis is required to recycle synaptic vesicles for fast and efficient neurotransmission. Amphiphysins are thought to be multiprotein adaptors that may contribute to this process by bringing together many of the proteins required for endocytosis. Their in vivo function, however, has yet to be determined.

Melatonin and melanocyte functions.

The effect of melatonin on melanocyte functions was studied by incubating whole-skin organ cultures with melatonin, as well as by assessing melatonin positivity in melanocytes versus dendricity and pigmentation, when arrested in the G(2) phase. From this study, it was observed that melatonin positivity is inversely related to the length of UV exposure. Increasing melatonin levels are related to decreasing dendricity and pigment donation during photoresponse in the G(2) phase.

Behavioral characterization and genetic analysis of the Drosophila melanogaster larval response to light as revealed by a novel individual assay.

A new assay was designed, named checker, that measures the individual response to light in the fruitfly Drosophila melanogaster larva. In this assay the Drosophila larva apparently modulates its pattern of locomotion when faced with a choice between a dark and lit environment by orienting its movement towards the dark environment. We show that, in this assay, a response to light can be measured as an increase in residence time in the dark versus the lit quadrant.

A simultaneous ex vivo model of embryogenesis: II. Vasculogenesis.

Vasculogenesis was simultaneously studied with embryogenesis in in ovo chick embryo culture, which was harvested at 40 hours. Endodermal cells and vascular endothelial cells were studied using a new combination of stains, immunohistochemistry (for nuclei and basement membrane) and NADPH-diaphorase activity in whole-mounts, paraffin sections and etched semithin sections. The model can be used for the study of developmental process of blood vessels as well as embryonic physiology of blood vessels vis-a-vis organogenesis in response to different angiogenic agents, drug trials, cancer therapy by angiostatic chemicals/radiations and toxins.

A simultaneous ex vivo model of embryogenesis: I. Organogenesis.

In vivo culture of chick embryo was carried out to develop an experimental interphase between in vitro and in vivo study of embryonic physiology. In the process, a simultaneous model of vasculogenesis and organogenesis has been worked out, which is impossible to achieve in mammalian system. Both early (40 hours of incubation) and late (64 & 88 hours of incubation) hours of cultures were conducted for morphological and morphometric studies.