Supramolecular Guest Exchange in Cucurbit[7]uril for Bioorthogonal Fluorogenic Imaging across the Visible Spectrum.

Fluorogenic probes that unmask fluorescence signals in response to bioorthogonal reactions are a powerful new addition to biological imaging. They can significantly reduce background fluorescence and minimize nonspecific signals, potentially enabling real-time, high-contrast imaging without the need to wash out excess fluorophores. While diverse classes of highly refined synthetic fluorophores are now readily available, integrating them into a bioorthogonal fluorogenic scheme still requires extensive design efforts and customized structural alterations to optimize quenching mechanisms for each specific fluorophore scaffold.

Reorganization of circadian activity and the pacemaker circuit under novel light regimes.

Many environmental features are cyclic, with predictable changes across the day, seasons and latitudes. Additionally, anthropogenic, artificial-light-induced changes in photoperiod or shiftwork-driven novel light/dark cycles also occur. Endogenous timekeepers or circadian clocks help organisms cope with such changes.

Drosophila melanogaster Neuromuscular Junction as a Model to Study Synaptopathies and Neuronal Autophagy.

Neuronal synapse dysfunction is a key characteristic of several neurodegenerative disorders, such as Alzheimer's disease, spinocerebellar ataxias, and Huntington's disease. Modeling these disorders to study synaptic dysfunction requires a robust and reproducible method for assaying the subtle changes associated with synaptopathies in terms of structure and function of the synapses. Drosophila melanogaster neuromuscular junctions (NMJs) serve as good models to study such alterations.

Restoration of Sleep and Circadian Behavior by Autophagy Modulation in Huntington's Disease.

Circadian and sleep defects are well documented in Huntington's disease (HD). Modulation of the autophagy pathway has been shown to mitigate toxic effects of mutant Huntingtin (HTT) protein. However, it is not clear whether autophagy induction can also rescue circadian and sleep defects.

A Subset of Circadian Neurons Expressing Enables Appropriate Phasing of Activity Rhythms in Under Warm Temperatures.

Under conditions of prolonged durations of warmth, flies counter potential temperature stress by shifting their locomotor activity from day into night when the conditions are likely to be less harsh. Modulation of a rhythmic behavior such as this in response to the environment would require interaction between at least 2 neuronal systems: (1) a system to receive input from the environment, and (2) the to correctly time rhythmic activity in response to this thermosensory input. Our previous studies found that a thermosensory mutant of the ion channel Transient Receptor Potential-A1 () failed to shift activity into the dark like control flies do and also identified the role of a specific cluster of the -expressing neurons, the neurons necessary for this.

A new player in circadian networks: Role of electrical synapses in regulating functions of the circadian clock.

Several studies have indicated that coherent circadian rhythms in behaviour can be manifested only when the underlying circadian oscillators function as a well-coupled network. The current literature suggests that circadian pacemaker neuronal networks rely heavily on communication mediated by chemical synapses comprising neuropeptides and neurotransmitters to regulate several behaviours and physiological processes. It has become increasingly clear that chemical synapses closely interact with electrical synapses and function together in the neuronal networks of most organisms.

Effects of Food Availability Cycles on Phase and Period of Activity-rest Rhythm in .

Foraging and feeding are indispensable for survival and their timing depends not only on the metabolic state of the animal but also on the availability of food resources in their environment. Since both these aspects are subject to change over time, these behaviors exhibit rhythmicity in occurrence. As the locomotor activity of an organism is related to its disposition to acquire food, and peak feeding in fruit flies has been shown to occur at a particular time of the day, we asked if cyclic food availability can entrain their rhythmic activity.

Populations Reared Under Tropical Semi-natural Conditions Evolve Season-dependent Differences in Timing of Eclosion.

Circadian clocks are considered an evolutionary adaptation to environmental cycles, helping organisms to adapt to daily and seasonal changes. However, most studies on the evolution of circadian rhythms have been carried out in controlled laboratory conditions; hence evolution of circadian clocks and rhythms in organisms reared under the influence of naturally varying time cues is not well understood. To address this, we reared large outbred fly populations in an outdoor enclosure on our institutional grounds in Bengaluru, southern India for about 150 generations, at the same time maintaining their ancestral control populations under standard laboratory conditions.

Glutamatergic Synapse Dysfunction in Neuromuscular Junctions Can Be Rescued by Proteostasis Modulation.

Glutamate is the major excitatory neurotransmitter in the nervous system, and the glutamatergic neuromuscular junctions (NMJs) offer a tractable platform to understand excitatory synapse biology both in health and disease. Synaptopathies are neurodegenerative diseases that are associated with synaptic dysfunction and often display compromised proteostasis. One such rare, progressive neurodegenerative condition, Spinocerebellar Ataxia Type 3 (SCA3) or Machado-Joseph Disease (MJD), is characterized by cerebellar ataxia, Parkinsonism, and degeneration of motor neuron synapses.

Hsp40 overexpression in pacemaker neurons delays circadian dysfunction in a Drosophila model of Huntington's disease.

Circadian disturbances are early features of neurodegenerative diseases, including Huntington's disease (HD). Emerging evidence suggests that circadian decline feeds into neurodegenerative symptoms, exacerbating them. Therefore, we asked whether known neurotoxic modifiers can suppress circadian dysfunction.

Under warm ambient conditions, Drosophila melanogaster suppresses nighttime activity via the neuropeptide pigment dispersing factor.

Rhythmic locomotor behaviour of flies is controlled by an endogenous time-keeping mechanism, the circadian clock, and is influenced by environmental temperatures. Flies inherently prefer cool temperatures around 25°C, and under such conditions, time their locomotor activity to occur at dawn and dusk. Under relatively warmer conditions such as 30°C, flies shift their activity into the night, advancing their morning activity bout into the early morning, before lights-ON, and delaying their evening activity into early night.

VANESSA-Shiny Apps for Accelerated Time-series Analysis and Visualization of Circadian Rhythm and Sleep Data.

Chronobiologists and sleep researchers often need to estimate various rhythm and sleep parameters from locomotor activity data from different organisms. The available open-source or expensive paid tools do not offer consolidated analysis and visualization options in one bundle, are often cumbersome for users unfamiliar with coding, offer very low customization options, introduce sources of human errors by requiring users to manually pick period and power values from periodogram plots, and do not generate reproducible reports. We present VANESSA, a family of cross-platform apps written in R, which, in our opinion, have several advantages compared with available tools-(a) open-source; (b) automatic period-power detection; (c) time-series filtering and smoothing; (d) high-resolution publication-quality figures with dynamic coloring, resizing, and light/dark shading; (e) reproducible code-report generation; (f) analysis and visualization of multiple monitor files, defining genotypes and replicates separately; and (g) sleep profile analysis, various sleep parameter estimations, quantification, bout analysis, and latency analysis.

Mechanosensory Stimulation via Expressing Neurons Can Induce Daytime Sleep in .

The neuronal and genetic bases of sleep, a phenomenon considered crucial for well-being of organisms, has been under investigation using the model organism Although sleep is a state where sensory threshold for arousal is greater, it is known that certain kinds of repetitive sensory stimuli, such as rocking, can indeed promote sleep in humans. Here we report that orbital motion-aided mechanosensory stimulation promotes sleep of male and female , independent of the circadian clock, but controlled by the homeostatic system. Mechanosensory receptor ()-expressing neurons in the chordotonal organs mediate this sleep induction: flies in which these neurons are either silenced or ablated display significantly reduced sleep induction on mechanosensory stimulation.

Gap junction protein Innexin2 modulates the period of free-running rhythms in .

A neuronal circuit of ∼150 neurons modulates rhythmic activity-rest behavior of . While it is known that coherent ∼24-hr rhythms in locomotion are brought about when 7 distinct neuronal clusters function as a network due to chemical communication amongst them, there are no reports of communication via electrical synapses made up of gap junctions. Here, we report that gap junction proteins, Innexins play crucial roles in determining the intrinsic period of activity-rest rhythms in flies.

Evidence for Co-Evolution of Masking With Circadian Phase in .

Heritable variation in the timing of rhythmic events with respect to daily time cues gives rise to chronotypes. Despite its importance, the mechanisms (clock or non-clock) regulating chronotypes remain elusive. Using artificial laboratory selection for divergent phasing of emergence of adults from pupae, our group has derived populations of which are and chronotypes for eclosion rhythm.

Responses of activity rhythms to temperature cues evolve in populations selected for divergent timing of eclosion.

Even though the rhythms in adult emergence and locomotor activity are two different phenomena that occur at distinct life stages of the fly life cycle, previous studies have hinted at similarities in certain aspects of the organisation of the circadian clock driving these two rhythms. For instance, the gene plays an important regulatory role in both rhythms. In an earlier study, we have shown that selection on timing of adult emergence behaviour in populations of leads to the co-evolution of temperature sensitivity of circadian clocks driving eclosion.

Waveform Plasticity under Entrainment to 12-h -cycles in : Behavior, Neuronal Network, and Evolution.

A crucial property of circadian clocks is the ability to regulate the shape of an oscillation over its cycle length (waveform) appropriately, thus enhancing Darwinian fitness. Many studies over the past decade have revealed interesting ways in which the waveform of rodent behavior could be manipulated, one of which is that the activity bout bifurcates under environments that have 2 light/dark cycles within one 24-h day (LDLD). It has been observed that such unique, although unnatural, environments reveal acute changes in the circadian clock network.

Dynamic host-guest interaction enables autonomous single molecule blinking and super-resolution imaging.

Synthetic host-guest complexes are inherently dynamic as they employ weak and reversible noncovalent interactions for their recognition processes. We strategically exploited dynamic supramolecular recognition between fluorescently labeled guest molecules to complementary cucurbit[7]uril hosts to obtain stochastic switching between fluorescence ON- and OFF-states, enabling PAINT-based nanoscopic imaging in cells and tissues.

Selection for Timing of Eclosion Results in Co-evolution of Temperature Responsiveness in .

Circadian rhythms in adult eclosion of are postulated to be regulated by a pair of coupled oscillators: one is the master clock that is light sensitive and temperature compensated and the other that is a slave oscillator whose period is temperature sensitive and whose phase is reflected in the overt behavior. Within this framework, we reasoned that in populations of that have been artificially selected for highly divergent phases of eclosion rhythm, there may be changes in this network of the master-slave oscillator system, via changes in the temperature-sensitive oscillator and/or the coupling of the light- and temperature-sensitive oscillators. We used light/dark cycles in conjunction with different constant ambient temperatures and 2 different amplitudes of temperature cycles in an overall cool or warm temperature and analyzed phases, gate width, and normalized amplitude of the rhythms in each of these conditions.

Accuracy of fruit-fly eclosion rhythms evolves by strengthening circadian gating rather than developmental fine-tuning.

Fruit flies () eclose from their pupae mainly around dawn. The timing of eclosion is thought to confer adaptive benefits to the organisms and thus shows remarkable accuracy. However, it is not clear what factors are involved in the evolution of such accuracy in natural populations.

RhythmicAlly: Your R and Shiny-Based Open-Source Ally for the Analysis of Biological Rhythms.

Research on circadian rhythms often requires researchers to estimate period, robustness/power, and phase of the rhythm. These are important to estimate, owing to the fact that they act as readouts of different features of the underlying clock. The commonly used tools, to this end, suffer from being very expensive, having very limited interactivity, being very cumbersome to use, or a combination of these.

Circadian Clock Properties and Their Relationships as a Function of Free-Running Period in Drosophila melanogaster.

The stability of circadian clock mechanisms under cyclic environments contributes to increased Darwinian fitness by accurately timing daily behavior and physiology. Earlier studies on biological clocks speculated that the timing of behavior and its accuracy are determined by the intrinsic period (τ) of the circadian clock under constant conditions, its stability, the period of the external cycle (T), and resetting of the clock by environmental time cues. However, most of these previous studies suffered from certain limitations, the major ones being a narrow range of examined τ values and a non-uniformity in the genetic background across the individuals tested.

Environmental cycles regulate development time via circadian clock mediated gating of adult emergence.

Background: Previous studies have implicated a role for circadian clocks in regulating pre-adult development of organisms. Among them two approaches are most notable: 1) use of insects whose clocks have different free-running periods and 2) imposition of artificial selection on either rate of development, timing of emergence or circadian period in laboratory populations. Using these two approaches, influence of clock on rate of development has been elucidated.

Synthetic Host-Guest Assembly in Cells and Tissues: Fast, Stable, and Selective Bioorthogonal Imaging via Molecular Recognition.

Bioorthogonal strategies are continuing to pave the way for new analytical tools in biology. Although a significant amount of progress has been made in developing covalent reaction based bioorthogonal strategies, balanced reactivity, and stability are often difficult to achieve from these systems. Alternatively, despite being kinetically beneficial, the development of noncovalent approaches that utilize fully synthetic and stable components remains challenging due to the lack of selectivity in conventional noncovalent interactions in the living cellular environment.

Wakefulness Is Promoted during Day Time by PDFR Signalling to Dopaminergic Neurons in Drosophila melanogaster.

Circadian clocks modulate timing of sleep/wake cycles in animals; however, the underlying mechanisms remain poorly understood. In , large ventral lateral neurons (l-LN) are known to promote wakefulness through the action of the neuropeptide pigment dispersing factor (PDF), but the downstream targets of PDF signalling remain elusive. In a screen using downregulation or overexpression (OEX) of the gene encoding PDF receptor (), we found that a subset of dopaminergic neurons responds to PDF to promote wakefulness during the day.