Takeo Katsuki

Establishing a Link Between Prescription Drug Abuse and Illicit Online Pharmacies: Analysis of Twitter Data

Background Youth and adolescent non-medical use of prescription medications (NUPM) has become a national epidemic. However, little is known about the association between promotion of NUPM behavior and access via the popular social media microblogging site, Twitter, which is currently used by a third of all teens. Objective In order to better assess NUPM behavior online, this study conducts surveillance and analysis of Twitter data to characterize the frequency of NUPM-related tweets and also identifies illegal access to drugs of abuse via online pharmacies. Methods Tweets were collected over a 2-week period from April 1-14, 2015, by applying NUPM keyword filters for both generic/chemical and street names associated with drugs of abuse using the Twitter public streaming application programming interface. Tweets were then analyzed for relevance to NUPM and whether they promoted illegal online access to prescription drugs using a protocol of content coding and supervised machine learning. Results A total of 2,417,662 tweets were collected and analyzed for this study. Tweets filtered for generic drugs names comprised 232,108 tweets, including 22,174 unique associated uniform resource locators (URLs), and 2,185,554 tweets (376,304 unique URLs) filtered for street names. Applying an iterative process of manual content coding and supervised machine learning, 81.72% of the generic and 12.28% of the street NUPM datasets were predicted as having content relevant to NUPM respectively. By examining hyperlinks associated with NUPM relevant content for the generic Twitter dataset, we discovered that 75.72% of the tweets with URLs included a hyperlink to an online marketing affiliate that directly linked to an illicit online pharmacy advertising the sale of Valium without a prescription. Conclusions This study examined the association between Twitter content, NUPM behavior promotion, and online access to drugs using a broad set of prescription drug keywords. Initial results are concerning, as our study found over 45,000 tweets that directly promoted NUPM by providing a URL that actively marketed the illegal online sale of prescription drugs of abuse. Additional research is needed to further establish the link between Twitter content and NUPM, as well as to help inform future technology-based tools, online health promotion activities, and public policy to combat NUPM online.

Flyception: imaging brain activity in freely walking fruit flies

Genetically encoded calcium sensors have enabled monitoring of neural activity in vivo using optical imaging techniques. Linking neural activity to complex behavior remains challenging, however, as most imaging systems require tethering the animal, which can impact the animals behavioral repertoire. Here, we report a method for monitoring the brain activity of untethered, freely walking Drosophila melanogaster during sensorially and socially evoked behaviors to facilitate the study of neural mechanisms that underlie naturalistic behaviors.

Jellyfish nervous systems

What are jellyfish? The term jellyfish generally refers to the umbrella-shaped gelatinous zooplanktons that belong to Scyphozoa (true jellyfish), Staurozoa (stalked jellyfish), Cubozoa (box jellyfish), and Hydrozoa of Phylum Cnidaria. Their sizes, shapes, and habitats are diverse. Sexually mature jellyfish range from millimeters to meters in diameter, and they can be found almost anywhere in the ocean from the arctic to the tropics and from the deep sea to the shoreline (some even live in freshwater).

Single mutations in sasA enable a simpler ΔcikA gene network architecture with equivalent circadian properties

Significance Complex cellular phenotypes are dependent upon a large underlying network of genes. Determining the number of interacting genes, the nature of their interactions, and the robustness of their encompassing network to cellular and genomic perturbations is important for understanding how genotype determines phenotype. Here, we approached these questions in the cyanobacterial circadian gene network by knocking out circadian input kinase A (cikA), a gene involved in synchronizing and relaying temporal signals within the cell, and screening for second-site suppressor mutations that rescue the severely altered cikA-null circadian phenotype. We identified two independent mutations in sasA (Synechococcus adaptive sensor A), a known clock-associated gene, that restore normal rhythms. These results change our conception of the circadian gene network architecture and show how easily the network can adapt to severe perturbations. The circadian input kinase of the cyanobacterium Synechococcus elongatus PCC 7942 (CikA) is important both for synchronizing circadian rhythms with external environmental cycles and for transferring temporal information between the oscillator and the global transcriptional regulator RpaA (regulator of phycobilisome-associated A). KOs of cikA result in one of the most severely altered but still rhythmic circadian phenotypes observed. We chemically mutagenized a cikA-null S. elongatus strain and screened for second-site suppressor mutations that could restore normal circadian rhythms. We identified two independent mutations in the Synechococcus adaptive sensor A (sasA) gene that produce nearly WT rhythms of gene expression, likely because they compensate for the loss of CikA on the temporal phosphorylation of RpaA. Additionally, these mutations restore the ability to reset the clock after a short dark pulse through an output-independent pathway, suggesting that SasA can influence entrainment through direct interactions with KaiC, a property previously unattributed to it. These experiments question the evolutionary advantage of integrating CikA into the cyanobacterial clock, challenge the conventional construct of separable input and output pathways, and show how easily the cell can adapt to restore phenotype in a severely compromised genetic network.

Developmental changes in expression, subcellular distribution, and function of Drosophila N-cadherin, guided by a cell-intrinsic program during neuronal differentiation

Cell adhesion molecules (CAMs) perform numerous functions during neural development. An individual CAM can play different roles during each stage of neuronal differentiation; however, little is known about how such functional switching is accomplished. Here we show that Drosophila N-cadherin (CadN) is required at multiple developmental stages within the same neuronal population and that its sub-cellular expression pattern changes between the different stages. During development of mushroom body neurons and motoneurons, CadN is expressed at high levels on growing axons, whereas expression becomes downregulated and restricted to synaptic sites in mature neurons. Phenotypic analysis of CadN mutants reveals that developing axons require CadN for axon guidance and fasciculation, whereas mature neurons for terminal growth and receptor clustering. Furthermore, we demonstrate that CadN downregulation can be achieved in cultured neurons without synaptic contact with other cells. Neuronal silencing experiments using Kir(2.1) indicate that neuronal excitability is also dispensable for CadN downregulation in vivo. Interestingly, downregulation of CadN can be prematurely induced by ectopic expression of a nonselective cation channel, dTRPA1, in developing neurons. Together, we suggest that switching of CadN expression during neuronal differentiation involves regulated cation influx within neurons.

Twitter-Based Detection of Illegal Online Sale of Prescription Opioid

Objectives To deploy a methodology accurately identifying tweets marketing the illegal online sale of controlled substances. Methods We first collected tweets from the Twitter public application program interface stream filtered for prescription opioid keywords. We then used unsupervised machine learning (specifically, topic modeling) to identify topics associated with illegal online marketing and sales. Finally, we conducted Web forensic analyses to characterize different types of online vendors. We analyzed 619 937 tweets containing the keywords codeine, Percocet, fentanyl, Vicodin, Oxycontin, oxycodone, and hydrocodone over a 5-month period from June to November 2015. Results A total of 1778 tweets (< 1%) were identified as marketing the sale of controlled substances online; 90% had imbedded hyperlinks, but only 46 were “live” at the time of the evaluation. Seven distinct URLs linked to Web sites marketing or illegally selling controlled substances online. Conclusions Our methodology can identify illegal online sale of prescription opioids from large volumes of tweets. Our results indicate that controlled substances are trafficked online via different strategies and vendors. Public Health Implications Our methodology can be used to identify illegal online sellers in criminal violation of the Ryan Haight Online Pharmacy Consumer Protection Act.

High-throughput and quantitative approaches for measuring circadian rhythms in cyanobacteria using bioluminescence.

The temporal measurement of a bioluminescent reporter has proven to be one of the most powerful tools for characterizing circadian rhythms in the cyanobacterium Synechococcus elongatus. Primarily, two approaches have been used to automate this process: (1) detection of cell culture bioluminescence in 96-well plates by a photomultiplier tube-based plate-cycling luminometer (TopCount Microplate Scintillation and Luminescence Counter, Perkin Elmer) and (2) detection of individual colony bioluminescence by iteratively rotating a Petri dish under a cooled CCD camera using a computer-controlled turntable. Each approach has distinct advantages. The TopCount provides a more quantitative measurement of bioluminescence, enabling the direct comparison of clock output levels among strains. The computer-controlled turntable approach has a shorter set-up time and greater throughput, making it a more powerful phenotypic screening tool. While the latter approach is extremely useful, only a few labs have been able to build such an apparatus because of technical hurdles involved in coordinating and controlling both the camera and the turntable, and in processing the resulting images. This protocol provides instructions on how to construct, use, and process data from a computer-controlled turntable to measure the temporal changes in bioluminescence of individual cyanobacterial colonies. Furthermore, we describe how to prepare samples for use with the TopCount to minimize experimental noise and generate meaningful quantitative measurements of clock output levels for advanced analysis.

Linking stimuli and behavior with fast near-whole brain recordings in adult Drosophila

Whole brain recordings give us a global perspective of the brain in action. In this study, we describe a method using light field microscopy to record near-whole brain calcium and voltage activity, at high speed, in behaving adult flies. We first obtained global activity maps for various stimuli and behaviors. Notably, we found that brain activity increased on a global scale when the fly walked but not when it groomed. This global increase with walking was particularly strong in dopamine neurons. Second, we extracted maps of spatially distinct sources of activity as well as their time series using principal component analysis and independent component analysis. The characteristic shapes in the maps matched the anatomy of sub-neuropil regions and in some cases a specific neuron type. Brain structures that responded to light and odor were consistent with previous reports, confirming the new technique’s validity. We also observed previously uncharacterized behavior-related activity, as well as patterns of spontaneous voltage activity.We provide a method to record near-whole brain activity in behaving adult flies and extract signals from specific anatomical structures. We image pan-neuronal calcium or voltage sensors fluorescence at high speed with light field microscopy. We then apply computational methods to extract functional maps, and find that their characteristic shape match the anatomy of sub-neuropile regions and sometimes small population of neurons. Associated time series are also consistent with the literature.We describe a method to record near-whole brain activity in behaving adult flies. Pan-neuronal calcium and voltage sensors were imaged at high speed with light field microscopy. Functional maps were then extracted by principal component analysis and independent component analysis. Their characteristic shapes match the anatomy of sub-neuropil regions and in some cases a specific neuron type. Responses to light and odor produced activity patterns consistent with previous techniques. Furthermore, the method detected activity linked to behavior as well as a previously uncharacterized pattern of spontaneous activity in the central complex.

Fast whole brain imaging in adult Drosophila during response to stimuli and behavior

Whole brain recordings give us a global perspective of the brain in action. In this study, we describe a method using light field microscopy to record near-whole brain calcium and voltage activity, at high speed, in behaving adult flies. We first obtained global activity maps for various stimuli and behaviors. Notably, we found that brain activity increased on a global scale when the fly walked but not when it groomed. This global increase with walking was particularly strong in dopamine neurons. Second, we extracted maps of spatially distinct sources of activity as well as their time series using principal component analysis and independent component analysis. The characteristic shapes in the maps matched the anatomy of sub-neuropil regions and in some cases a specific neuron type. Brain structures that responded to light and odor were consistent with previous reports, confirming the new technique’s validity. We also observed previously uncharacterized behavior-related activity, as well as patterns of spontaneous voltage activity.We provide a method to record near-whole brain activity in behaving adult flies and extract signals from specific anatomical structures. We image pan-neuronal calcium or voltage sensors fluorescence at high speed with light field microscopy. We then apply computational methods to extract functional maps, and find that their characteristic shape match the anatomy of sub-neuropile regions and sometimes small population of neurons. Associated time series are also consistent with the literature.We describe a method to record near-whole brain activity in behaving adult flies. Pan-neuronal calcium and voltage sensors were imaged at high speed with light field microscopy. Functional maps were then extracted by principal component analysis and independent component analysis. Their characteristic shapes match the anatomy of sub-neuropil regions and in some cases a specific neuron type. Responses to light and odor produced activity patterns consistent with previous techniques. Furthermore, the method detected activity linked to behavior as well as a previously uncharacterized pattern of spontaneous activity in the central complex.

Activity sources from fast large-scale brain recordings in adult Drosophila

Whole brain recordings give us a global perspective of the brain in action. In this study, we describe a method using light field microscopy to record near-whole brain calcium and voltage activity, at high speed, in behaving adult flies. We first obtained global activity maps for various stimuli and behaviors. Notably, we found that brain activity increased on a global scale when the fly walked but not when it groomed. This global increase with walking was particularly strong in dopamine neurons. Second, we extracted maps of spatially distinct sources of activity as well as their time series using principal component analysis and independent component analysis. The characteristic shapes in the maps matched the anatomy of sub-neuropil regions and in some cases a specific neuron type. Brain structures that responded to light and odor were consistent with previous reports, confirming the new technique’s validity. We also observed previously uncharacterized behavior-related activity, as well as patterns of spontaneous voltage activity.We provide a method to record near-whole brain activity in behaving adult flies and extract signals from specific anatomical structures. We image pan-neuronal calcium or voltage sensors fluorescence at high speed with light field microscopy. We then apply computational methods to extract functional maps, and find that their characteristic shape match the anatomy of sub-neuropile regions and sometimes small population of neurons. Associated time series are also consistent with the literature.We describe a method to record near-whole brain activity in behaving adult flies. Pan-neuronal calcium and voltage sensors were imaged at high speed with light field microscopy. Functional maps were then extracted by principal component analysis and independent component analysis. Their characteristic shapes match the anatomy of sub-neuropil regions and in some cases a specific neuron type. Responses to light and odor produced activity patterns consistent with previous techniques. Furthermore, the method detected activity linked to behavior as well as a previously uncharacterized pattern of spontaneous activity in the central complex.