Cell quantification assays are essential components of most biological and clinical labs. However, many currently available quantification assays, including flow cytometry and commercial cell counting systems, suffer from unique drawbacks that limit their overall efficacy. In order to address the shortcomings of traditional quantification assays, we have designed a robust, low-cost, automated microscopy-based cytometer that quantifies individual cells in a multiwell plate using tools readily available in most labs. Plating and subsequent quantification of various dilution series using the automated microscopy-based cytometer demonstrates the single-cell sensitivity, near-perfect R2 accuracy, and greater than 5-log dynamic range of our system. Further, the microscopy-based cytometer is capable of obtaining absolute counts of multiple cell types in one well as part of a co-culture setup. To demonstrate this ability, we recreated an experiment that assesses the tumoricidal properties of primed macrophages on co-cultured tumor cells as a proof-of-principle test. The results of the experiment reveal that primed macrophages display enhanced cytotoxicity toward tumor cells while simultaneously losing the ability to proliferate, an example of a dynamic interplay between two cell populations that our microscopy-based cytometer is successfully able to elucidate.

22Cancer has proven to be an extremely difficult challenge to treat. Several 23 fundamental issues currently underlie cancer treatment including differentiating 24 self from non-self, functional coupling of the recognition and therapeutic 25 components of various therapies, and the propensity of cancerous cells to develop 26 resistance to common treatment modalities via evolutionary pressure. Given these 27 limitations, there is an increasing need to develop an all-encompassing therapeutic 28 that can uniquely target malignant cells, decouple recognition from treatment, and 29 overcome evolutionarily driven cancer resistance. We describe herein, a new class 30 of programmable self-assembling dsRNA-based cancer therapeutics, that uniquely 31 targets aberrant genetic sequences, and in a functionally decoupled manner, 32 undergoes oncogenic RNA activated displacement (ORAD), initiating a therapeutic 33 cascade that induces apoptosis and immune activation. As a proof-of-concept, we 34show that RNA strands targeting the EWS/Fli1 fusion gene in Ewing Sarcoma cells 35 that are end-blocked with phosphorothioate bonds and additionally sealed with a 36 2'-U modified DNA protector can be used to induce specific and potent killing of cells 37 containing the target oncogenic sequence, but not wildtype. 38 39 40 41 42

Cell quantification assays are essential components of most biological and clinical labs. However, many currently available quantification assays, including flow cytometry and commercial cell counting systems, suffer from unique drawbacks that limit their overall efficacy. In order to address the shortcomings of traditional quantification assays, we have designed a robust, low-cost, automated optical cell cytometer that quantifies individual cells in a multiwell plate using tools readily available in most labs. Plating and subsequent quantification of various dilution series using the automated optical cytometer demonstrates the single-cell sensitivity, near-perfect R2 accuracy, and greater than 5-log dynamic range of our system. Further, the optical cytometer is capable of obtaining absolute counts of multiple cell types in one well as part of a co-culture setup. To demonstrate this ability, we recreated an experiment that assesses the tumoricidal properties of primed macrophages on co-cultured tumor cells as a proof-of-principle test. The results of the experiment reveal that primed macrophages display enhanced cytotoxicity towards tumor cells while simultaneously losing the ability to proliferate, an example of a dynamic interplay between two cell populations that our optical cytometer is successfully able to elucidate.

A 23-year-old male presented to the hospital with altered mental status (AMS) and hypoglycemia requiring admission to the ICU. He had improvement in AMS after administration of dextrose 50% and naloxone and endorsed the use of alcohol, cocaine, and marijuana that morning. It was confirmed with a positive urine toxicology screen for cocaine and tetrahydrocannabinol (THC). During this hospital admission, his physical examination was notable for paraplegia with no motor abilities from the T6 dermatome and below. Sensation was intact throughout all dermatomes but he was found to have urinary retention. Workup included an abnormal MRI showing T2 signal spanning from T2-T8, raising a high suspicion of a probable acute ischemic spinal cord infarction. Several hours after admission, the patient began to exhibit the first signs of abnormal bowel function and experienced one episode of hematemesis, prolonging his ICU stay.