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Chatroulette enables you to share your thoughts or experiences with a new random person who doesn't know anything about you and whom you do not know. Understand this before it's too late: When the educational products are "watch live sex free," it's our children who are the products -- or the prey. In one embodiment, the toxicity, e.g., in vivo acute neurotoxicity, of the molecule is tested by measuring intracellular free calcium oscillations (calcium oscillations) in vitro in neuronal cells which are in contact or have been in contact with the molecule. In some embodiments, fluorescence of the stained cells (including stained individual cells) can be measured by confocal, or standard, fluorescence microscopy, optionally at a number of time points or continuously (e.g., real-time) to provide, for example, time lapse measurements. Calcium oscillations are important for the proper functions of neuronal cells. In certain embodiments, molecules that cause greater than desired reductions in calcium oscillations are considered to be molecules that have unacceptable neurotoxicity. In other embodiments, the calcium oscillations measured in the present methods are the combination of the oscillation frequency and magnitude.



In some embodiments, the behavioral score is measured by the following methods, including a novel object rejection test, a water maze test, a gait analysis test, and/or any combination thereof. Besides King's boss, other people have started to come to her defense, including Senator Cory Booker and NBC morning show host Willie Geist. But the singer has decided to show fans she will not take the criticism without commenting back. Try cooking for her and show her your culinary skills from time to time! In one embodiment, the calcium oscillations measured in the present methods are the cumulative increase in calcium oscillations within a culture of neuronal cells, whereby the time to reach the maximum fluorescence signal constitutes the magnitude of the calcium response. The term "oscillation frequency" refers to the time between oscillations. For example, long term toxicities can be determined by measuring the change in tubulin intensity in a cell by the molecule when the cell comes in contact with a molecule.



In certain embodiments, the change in tubulin intensity is measured along with one or both of the change in calcium oscillation, sequence score, and/or in vivo tolerability assay. Any other methods disclosed in this application and/or known in the art can further be combined with the calcium oscillation assay and/or the sequence score method. In some embodiments, the calcium oscillations are NMDA-dependent calcium oscillations. Calcium oscillations can also regulate interactions of neurons with associate glia, in addition to other associated neurons in the network, to release other neurotransmitters in addition to glutamate. In other embodiments, an in vivo tolerability study can be used in combination with the calcium oscillation assay and/or the sequence score method. The disclosure also provides an in vivo tolerability assay that can be used separately or combined with the calcium oscillation assay and/or the sequence score method. In one aspect, the disclosure sets forth a calcium oscillation assay as one way of measuring or predicting toxicity of a molecule.



In some embodiments, the calcium oscillations of a molecule having intolerable in vivo neurotoxicity are less than 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the calcium oscillations in a vehicle control cell. EC50 is the median effective concentration of a therapeutic molecule relative to a vehicle or saline control. In another embodiment, a molecule of the invention targets one or more endogenously produced proteins or peptides in vivo, one or more mRNAs or pre-mRNAs encoding the proteins or peptides, or one or more genes encoding the proteins or peptides. In one embodiment, a behavioral score is obtained with a novel object recognition test. In one embodiment, a behavioral score is obtained with a gait analysis test, such as the Catwalk. In one embodiment, the method comprises a behavioral test score, which can be measured by administering the molecule to a mammal and grading the mammal's behavioral performance.