CODEX, a neural network approach to explore signaling dynamics landscapes.

Current studies of cell signaling dynamics that use live cell fluorescent biosensors routinely yield thousands of single-cell, heterogeneous, multi-dimensional trajectories. Typically, the extraction of relevant information from time series data relies on predefined, human-interpretable features. Without a priori knowledge of the system, the predefined features may fail to cover the entire spectrum of dynamics. Here we present CODEX, a data-driven approach based on convolutional neural networks (CNNs) that identifies patterns in time series. It does not require a priori information about the biological system and the insights into the data are built through explanations of the CNNs' predictions. CODEX provides several views of the data: visualization of all the single-cell trajectories in a low-dimensional space, identification of prototypic trajectories, and extraction of distinctive motifs. We demonstrate how CODEX can provide new insights into ERK and Akt signaling in response to various growth factors, and we recapitulate findings in p53 and TGFß-SMAD2 signaling.

Ionizing radiation reduces larval brain size by inducing premature differentiation of Drosophila neural stem cells.

DNA damaging agents, such as ionizing radiation (IR), induce cell cycle arrest, senescence, differentiation, or cell death of stem cells, which may affect tissue homeostasis. The specific response of stem cells upon irradiation seems to vary depending on the cell type and their developmental stages. Drosophila larval brain contains neural stem cells called neuroblasts (NBs) and maintaining an appropriate number of NBs is critical to maintain brain size. Irradiation of larvae at early larval stage results in microcephaly, whereas the DNA damage response of NBs that could explain this small brain size is not clearly understood. We observed that the irradiation of larvae in the second instar retarded brain growth, accompanied by fewer NBs. The IR-induced microcephaly does not seem to result from apoptosis since the irradiated larval brain was not stained with activated Caspase nor was the microcephaly affected by the ectopic expression of the apoptosis inhibitor. When analyzed for the percentage of mitotic cells, irradiated NBs recovered their proliferative potential within 6 h post-irradiation after transient cell cycle arrest. However, IR eventually reduced the proliferation of NBs at later time points and induced the premature differentiation of NBs. In summary, IR-induced microcephaly occurs by NB loss due to premature differentiation, rather than apoptotic cell death.

Role of p53 isoforms in the DNA damage response during Drosophila oogenesis.

The tumor suppressor p53 is involved in the DNA damage response and induces cell cycle arrest or apoptosis upon DNA damage. Drosophila p53 encodes two isoforms, p53A and p53B, that induce apoptosis in somatic cells. To investigate the roles of Drosophila p53 isoforms in female germline cells, the DNA damage response was analyzed in the adult ovary. Early oogenesis was sensitive to irradiation and lok-, p53-, and hid-dependent cell death occurred rapidly after both low- and high-dose irradiation. Both p53 isoforms were responsible for this cell death. On the other hand, delayed cell death in mid-oogenesis was induced at a low level only after high-dose irradiation in a p53-independent manner. The daily egg production, which did not change after low-dose irradiation, was severely reduced after high-dose irradiation in p53 mutant females due to the loss of germline stem cells. When the p53A or p53B isoform was expressed in the germline cells in the p53 mutant females at levels that do not affect normal oogenesis, p53A, but not p53B, restored the fertility of the irradiated female. In summary, moderate expression of p53A is critical to maintain the function of germline stem cells during normal oogenesis as well as after high-dose irradiation.

Selective Killing of RAS-Malignant Tissues by Exploiting Oncogene-Induced DNA Damage.

Several oncogenes induce untimely entry into S phase and alter replication timing and progression, thereby generating replicative stress, a well-known source of genomic instability and a hallmark of cancer. Using an epithelial model in Drosophila, we show that the RAS oncogene, which triggers G1/S transition, induces DNA damage and, at the same time, silences the DNA damage response pathway. RAS compromises ATR-mediated phosphorylation of the histone variant H2Av and ATR-mediated cell-cycle arrest in G2 and blocks, through ERK, Dp53-dependent induction of cell death. We found that ERK is also activated in normal tissues by an exogenous source of damage and that this activation is necessary to dampen the pro-apoptotic role of Dp53. We exploit the pro-survival role of ERK activation upon endogenous and exogenous sources of DNA damage to present evidence that its genetic or chemical inhibition can be used as a therapeutic opportunity to selectively eliminate RAS-malignant tissues.

Non-mammalian Hosts and Photobiomodulation: Do All Life-forms Respond to Light?

Photobiomodulation (PBM), also known as low-level laser (light) therapy, was discovered over 50 years ago, but only recently has it been making progress toward wide acceptance. PBM originally used red and near-infrared (NIR) lasers, but now other wavelengths and non-coherent light-emitting diodes (LEDs) are being explored. The almost complete lack of side effects makes the conduction of controlled clinical trials relatively easy. Laboratory research has mainly concentrated on mammalian cells (normal or cancer) in culture, and small rodents (mice and rats) as models of different diseases. A sizeable body of work was carried out in the 1970s and 1980s in Russia looking at various bacterial and fungal cells. The present review covers some of these studies and a recent number of papers that have applied PBM to so-called "model organisms." These models include flies (Drosophila), worms (Caenorhabditis elegans), fish (zebrafish) and caterpillars (Galleria). Much knowledge about the genomics and proteomics, and many reagents for these organisms already exist. They are inexpensive to work with and have lower regulatory barriers compared to vertebrate animals. Other researchers have studied different models (snails, sea urchins, Paramecium, toads, frogs and chickens). Plants may respond to NIR light differently from visible light (photosynthesis and photomorphogenesis) but PBM in plants has not been much studied. Veterinarians routinely use PBM to treat non-mammalian patients. The conclusion is that red or NIR light does indeed have significant biologic effects conserved over many different kingdoms, and perhaps it is true that "all life-forms respond to light."

Irradiation dose response under hypoxia for the application of the sterile insect technique in Drosophila suzukii.

Treating insects with a lower oxygen atmosphere before and during exposure to radiation can mitigate some of the negative physiological effects due to the irradiation. The irradiation of pupae under oxygen-reduced environment such as hypoxia or anoxia is routinely used in the sterile insect technique (SIT) of some tephritid species as it provides radiological protection. This treatment allows to have the sterile pupae already in sealed containers facilitating the shipment. SIT is an environment friendly control tactic that could be used to manage populations of Drosophila suzukii in confined areas such as greenhouses. The objectives of this study were to assess the effect of irradiation on the reproductive sterility in D. suzukii males and females under low-oxygen atmosphere (hypoxia) and atmosphere conditions (normoxia). Additionally, we assessed the differences in radiological sensitivity of pupae treated under hypoxia and normoxia conditions. Finally, the effect on emergence rate and flight ability of the irradiated D. suzukii adults exposed to doses that induced >99% of sterility were assessed. Pupae needed a 220 Gy irradiation dose to achieve >99% of egg hatch sterility in males irrespective of the atmosphere condition. For females the same level of sterility was achieved already at 75 Gy and 90 Gy for the normoxia and hypoxia treatments, respectively. Radiation exposure at 170 and 220 Gy under the two atmosphere treatments did not have any effect on the emergence rate and flight ability of D. suzukii males and females. Therefore, hypoxia conditions can be used as part of an area-wide insect pest management program applying SIT to facilitate the protocols of packing, irradiation and shipment of sterile D. suzukii pupae.

Ionizing radiation induces stem cell-like properties in a caspase-dependent manner in Drosophila.

Cancer treatments including ionizing radiation (IR) can induce cancer stem cell-like properties in non-stem cancer cells, an outcome that can interfere with therapeutic success. Yet, we understand little about what consequences of IR induces stem cell like properties and why some cancer cells show this response but not others. In previous studies, we identified a pool of epithelial cells in Drosophila larval wing discs that display IR-induced stem cell-like properties. These cells are resistant to killing by IR and, after radiation damage, change fate and translocate to regenerate parts of the disc that suffered more cell death. Here, we report the identification of two new pools of cells with IR-induced regenerative capability. We addressed how IR exposure results in the induction of stem cell-like behavior, and found a requirement for IR-induced caspase activity and for Zfh2, a transcription factor and an effector in the JAK/STAT pathway. Unexpectedly, the requirement for caspase activity was cell-autonomous within cell populations that display regenerative behavior. We propose a model in which the requirement for caspase activity and Zfh2 can be explained by apoptotic and non-apoptotic functions of caspases in the induction of stem cell-like behavior.

Muller's nobel prize research and peer review.

BACKGROUND: This paper assesses possible reasons why Hermann J. Muller avoided peer-review of data that became the basis of his Nobel Prize award for producing gene mutations in male Drosophila by X-rays. METHODS: Extensive correspondence between Muller and close associates and other materials were obtained from preserved papers to compliment extensive publications by and about Muller in the open literature. These were evaluated for potential historical insights that clarify why he avoided peer-review of his Nobel Prize findings. RESULTS: This paper clarifies the basis of Muller's (Muller HJ, Sci 66 84-87, 1927c) belief that he produced X-ray induced "gene" mutations in Drosophila. It then shows his belief was contemporaneously challenged by his longtime friend/confidant and Drosophila geneticist, Edgar Altenburg. Altenburg insisted that Muller may have simply poked large holes in chromosomes with massive doses of X-rays, and needed to provide proof of gene "point" mutations. Given the daunting and uncertain task to experimentally address this criticism, especially within the context of trying to become first to produce gene mutations, it is proposed that Muller purposely avoided peer-review while rushing to publish his paper in Science to claim discovery primacy without showing any data. The present paper also explores ethical issues surrounding these actions, including those of the editor of Science, James McKeen Catell and Altenburg, and their subsequent impact on the scientific and regulatory communities. CONCLUSION: This historical analysis suggests that Muller deliberately avoided peer-review on his most significant findings because he was extremely troubled by the insightful and serious criticism of Altenburg, which suggested he had not produced gene mutations as he claimed. Nonetheless, Muller manipulated this situation (i.e., publishing a discussion within Science with no data, publishing a poorly written non-peer reviewed conference proceedings with no methods and materials, and no references) due to both the widespread euphoria over his claim of gene mutation and confidence that Altenburg would not publically challenge him. This situation permitted Muller to achieve his goal to be the first to produce gene mutations while buying him time to later try to experimentally address Altenburg's criticisms, and a possible way to avoid discovery of his questionable actions.

The circadian activity rhythm is reset by nanowatt pulses of ultraviolet light.

The circadian pacemaker synchronizes to the Earth's rotation by tracking step-by-step changes in illumination that occur as the sun passes the horizon. While twilight progressions of irradiance and colour are considered important stimuli in this process, comparably less thought has been given to the possibility that ultraviolet A (UVA) radiation might actually play a more formative role given its evolutionary significance in shaping 24 h timekeeping. Here, we show that Drosophila activity rhythms can be phase-shifted by UVA light at an energy range seated well below that of the visible spectrum. Because the energy threshold for this resetting matches the incident amount of UVA on the human retina at twilight, our results suggest that UVA light has the potential to function as a similar time cue in people.

Epithelial cell shape change of Drosophila as a biomonitoring model for the dose assessment of environmental radiation.

Inevitable exposure to ionizing radiation from natural and human-made sources has been increasing over time. After nuclear disasters, such as the Fukushima accident, the public concerns on health risk of radiation exposure because of radioactive contamination of the environment have increased. However, it is very difficult to assess the biological effects of exposure caused by environmental radiation. A reliable and rapid bioassay to monitor the physiological effects of radiation exposure is therefore needed. Here, we quantitatively analyzed the changes in cell shape in Drosophila epidermis after irradiation as a model for biomonitoring of radiation. Interestingly, the number of irregularly shaped epithelial cells was increased by irradiation in a dose-dependent manner. A dose-response curve constructed with the obtained data suggests that the measurement of the number of irregular shaped cell in the epidermis is useful for the assessment of radiation dose. In addition, a comparison of the variation in the different samples and the data scored by different observers showed that our evaluation for cellular morphology was highly reliable and accurate and would, therefore, have immense practical application. Overall, our study suggests that detection of morphological changes in the epithelial cells is one of the efficient ways to quantify the levels of exposure to radioactive radiation from the environment.

Circadian clock activity of cryptochrome relies on tryptophan-mediated photoreduction.

Cryptochromes (CRYs) entrain the circadian clocks of plants and animals to light. Irradiation of the Drosophila cryptochrome (dCRY) causes reduction of an oxidized flavin cofactor by a chain of conserved tryptophan (Trp) residues. However, it is unclear how redox chemistry within the Trp chain couples to dCRY-mediated signaling. Here, we show that substitutions of four key Trp residues to redox-active tyrosine and redox-inactive phenylalanine tune the light sensitivity of dCRY photoreduction, conformational activation, cellular stability, and targeted degradation of the clock protein timeless (TIM). An essential surface Trp gates electron flow into the flavin cofactor, but can be relocated for enhanced photoactivation. Differential effects of Trp-mediated flavin photoreduction on cellular turnover of TIM and dCRY indicate that these activities are separated in time and space. Overall, the dCRY Trp chain has evolutionary importance for light sensing, and its manipulation has implications for optogenetic applications of CRYs.

Effects of Irradiation Dose on Sterility Induction and Quality Parameters of Drosophila suzukii (Diptera: Drosophilidae).

Drosophila suzukii (Matsumura, 1931) (Diptera: Drosophilidae) is a widely distributed pest of soft-skinned and stone fruits that is controlled mainly with pesticides. An alternative to the chemical control is the sterile insect technique (SIT), an ecologically friendly method of pest management that could be used against D. suzukii. The objective of the present study was to evaluate the effects of gamma radiation on reproductive sterility, ovarian morphometry, and quality parameters of D. suzukii. Full female sterility was achieved at 75 Gy, while an adequate level of male sterility (99.67%) was obtained at 200 Gy. The ovarian size showed an exponential decay in function of irradiation dose increase. There was no significant influence of irradiation dose on the quality parameters evaluated. Our data suggest that gamma radiation can be recommended to be used in an SIT program for D. suzukii.

Effect of Low-Oxygen Conditions Created by Modified Atmosphere Packaging on Radiation Tolerance in Drosophila suzukii (Diptera: Drosophilidae) in Sweet Cherries.

Modified atmosphere packaging (MAP) creates a low-oxygen (O2) environment that can increase the shelf life of fresh produce by decreasing respiration and the growth of pathogens. Low oxygen may also increase insect tolerance to irradiation (IR), and the use of MAP with products treated by IR to control quarantine pests before export may inadvertently compromise treatment efficacy. Spotted wing drosophila, Drosophila suzukii Matsumura (Diptera: Drosophilidae) is a quarantine pest of stone and small fruits and a potential target for postharvest IR treatment. The effect of low oxygen generated by MAP at ambient temperatures on the radiation tolerance of D. suzukii infesting sweet cherries was examined. Early pupal stage D. suzukii were inserted into ripe sweet cherries and treated by 1) MAP + IR, 2) IR alone, 3) MAP alone, or 4) no MAP and no IR and held for adult emergence. Three types of commercially available MAP products were tested that produced different oxygen concentrations between 3 and 15%, and a sublethal radiation dose (60 Gy) was used to allow comparisons between the treatments. Xtend PP61 bags (3.2-4.8% O2), Xtend PP71 bags (5.4-8.6% O2), and Xtend PP53 bags (13.6-15.4% O2) did not enhance survivorship to the adult stage in D. suzukii pupae irradiated at 60 Gy in sweet cherries. MAP use should not compromise phytosanitary IR treatment against D. suzukii in exported sweet cherries or other fruit.

Exploratory search during directed navigation in C. elegans and Drosophila larva.

Many organisms-from bacteria to nematodes to insect larvae-navigate their environments by biasing random movements. In these organisms, navigation in isotropic environments can be characterized as an essentially diffusive and undirected process. In stimulus gradients, movement decisions are biased to drive directed navigation toward favorable environments. How does directed navigation in a gradient modulate random exploration either parallel or orthogonal to the gradient? Here, we introduce methods originally used for analyzing protein folding trajectories to study the trajectories of the nematode Caenorhabditis elegans and the Drosophila larva in isotropic environments, as well as in thermal and chemical gradients. We find that the statistics of random exploration in any direction are little affected by directed movement along a stimulus gradient. A key constraint on the behavioral strategies of these organisms appears to be the preservation of their capacity to continuously explore their environments in all directions even while moving toward favorable conditions.

Mapping Quantitative Trait Loci Underlying Circadian Light Sensitivity in Drosophila.

Despite the significant advance in our understanding of the molecular basis of light entrainment of the circadian clock in Drosophila, the underlying genetic architecture is still largely unknown. The aim of this study was to identify loci associated with variation in circadian photosensitivity, which are important for the evolution of this trait. We have used complementary approaches that combined quantitative trait loci (QTL) mapping, complementation testing, and transcriptome profiling to dissect this variation. We identified a major QTL on chromosome 2, which was subsequently fine mapped using deficiency complementation mapping into 2 smaller regions spanning 139 genes, some of which are known to be involved in functions that have been previously implicated in light entrainment. Two genes implicated with the clock and located within that interval, timeless and cycle, failed to complement the QTL, indicating that alleles of these genes contribute to the variation in light response. Specifically, we find that the timeless s/ ls polymorphism that has been previously shown to constitute a latitudinal cline in Europe is also segregating in our recombinant inbred lines and is contributing to the phenotypic variation in light sensitivity. We also profiled gene expression in 2 recombinant inbred strains that differ significantly in their photosensitivity and identified a total of 368 transcripts that showed differential expression (false discovery rate < 0.1). Of 131 transcripts that showed a significant recombinant inbred line by treatment interaction (i.e., putative expression QTL), 4 are located within QTL2.

The sterile insect technique for the management of the spotted wing drosophila, Drosophila suzukii: Establishing the optimum irradiation dose.

The spotted wing drosophila Drosophila suzukii Matsumura (Diptera: Drosophilidae), a pest of berries stone fruits, invaded North America and Europe in 2008. Current control methods rely mainly on insecticides. The sterile insect technique (SIT) has potential as an additional control tactic for the integrated management of D. suzukii. As a step towards the development of the SIT, this study aimed at finding the optimum irradiation dose to sterilize D. suzukii under controlled laboratory conditions. Four-day-old D. suzukii pupae were irradiated 12 to 24 hours prior to adult emergence in a 60Co Gamma Cell 220 and in a 137Cs Gamma Cell 3000 with doses of 30, 50, 70, 80, 90, 100 or 120 Gy. Emergence rate (88.1%), percent of deformed flies (4.0%) and survival curves were not affected by the tested irradiation doses. However, some reproductive parameters of the flies were affected by irradiation. Females irradiated with a dose of 50 Gy or more had almost no fecundity. When non-irradiated females were mated with irradiated males, egg hatch decreased exponentially with irradiation dose from 82.6% for the untreated control males to 4.0% for males irradiated with 120 Gy. Mortality of F1 individuals from the irradiated treatment also occurred during larval and pupal stages, with an egg to adult survival of 0.2%. However, descendants produced by the irradiated generation were fertile. These results are an encouraging first experimental step towards the development of the SIT for the management of D. suzukii populations.

Turns with multiple and single head cast mediate Drosophila larval light avoidance.

Drosophila larvae exhibit klinotaxis when placed in a gradient of temperature, chemicals, or light. The larva samples environmental stimuli by casting its head from side to side. By comparing the results of two consecutive samples, it decides the direction of movement, appearing as a turn proceeded by one or more head casts. Here by analyzing larval behavior in a light-spot-based phototaxis assay, we showed that, in addition to turns with a single cast (1-cast), turns with multiple head casts (n-cast) helped to improve the success of light avoidance. Upon entering the light spot, the probability of escape from light after the first head cast was only ~30%. As the number of head casts increased, the chance of successful light avoidance increased and the overall chance of escaping from light increased to >70%. The amplitudes of first head casts that failed in light avoidance were significantly smaller in n-cast turns than those in 1-cast events, indicating that n-cast turns might be planned before completion of the first head cast. In n-casts, the amplitude of the second head cast was generally larger than that of the first head cast, suggesting that larvae tried harder in later attempts to improve the efficacy of light avoidance. We propose that both 1-cast turns and n-cast turns contribute to successful larval light avoidance, and both can be initiated at the first head cast.

Flaws in the LNT single-hit model for cancer risk: An historical assessment.

The LNT single-hit model was derived from the Nobel Prize-winning research of Herman J. Muller who showed that x-rays could induce gene mutations in Drosophila and that the dose response for these so-called mutational events was linear. Lewis J. Stadler, another well-known and respected geneticist at the time, strongly disagreed with and challenged Muller's claims. Detailed evaluations by Stadler over a prolonged series of investigations revealed that Muller's experiments had induced gross heritable chromosomal damage instead of specific gene mutations as had been claimed by Muller at his Nobel Lecture. These X-ray-induced alterations became progressively more frequent and were of larger magnitude (more destructive) with increasing doses. Thus, Muller's claim of having induced discrete gene mutations represented a substantial speculative overreach and was, in fact, without proof. The post hoc arguments of Muller to support his gene mutation hypothesis were significantly challenged and weakened by a series of new findings in the areas of cytogenetics, reverse mutation, adaptive and repair processes, and modern molecular methods for estimating induced genetic damage. These findings represented critical and substantial limitations to Muller's hypothesis of X-ray-induced gene mutations. Furthermore, they challenged the scientific foundations used in support of the LNT single-hit model by severing the logical nexus between Muller's data on radiation-induced inheritable alterations and the LNT single-hit model. These findings exposed fundamental scientific flaws that undermined not only the seminal recommendation of the 1956 BEAR I Genetics Panel to adopt the LNT single-hit Model for risk assessment but also any rationale for its continued use in the present day.

The role of PDF neurons in setting the preferred temperature before dawn in Drosophila.

Animals have sophisticated homeostatic controls. While mammalian body temperature fluctuates throughout the day, small ectotherms, such as Drosophila achieve a body temperature rhythm (BTR) through their preference of environmental temperature. Here, we demonstrate that pigment dispersing factor (PDF) neurons play an important role in setting preferred temperature before dawn. We show that small lateral ventral neurons (sLNvs), a subset of PDF neurons, activate the dorsal neurons 2 (DN2s), the main circadian clock cells that regulate temperature preference rhythm (TPR). The number of temporal contacts between sLNvs and DN2s peak before dawn. Our data suggest that the thermosensory anterior cells (ACs) likely contact sLNvs via serotonin signaling. Together, the ACs-sLNs-DN2s neural circuit regulates the proper setting of temperature preference before dawn. Given that sLNvs are important for sleep and that BTR and sleep have a close temporal relationship, our data highlight a possible neuronal interaction between body temperature and sleep regulation.