ABSTRACT
The pupil constricts or dilates in response to a luminance increase or decrease, and these transient pupillary responses are controlled by the parasympathetic and sympathetic pathways. Although pupillary responses of the two eyes are highly correlated, they are not always identical (referred to as anisocoria). For example, there are unequal direct and consensual pupillary constriction responses after an increase in luminance to one eye. While contraction anisocoria (i.e. constriction) has been demonstrated in the pupillary light reflex, it is not yet known if there is also dilation anisocoria in the pupillary darkness reflex. Unlike previous studies that focused on the pupillary light reflex, we examined response anisocoria in both pupillary light and darkness reflexes. While requiring participants to maintain central fixation, we presented a light or dark stimulus to either the right or left visual field to induce transient pupillary constriction or dilation. Both the pupillary light and darkness reflexes had significantly larger ipsilateral responses compared to the contralateral responses relative to the stimulated visual field. The observed ipsilateral effects occurred significantly faster in the light than darkness reflex, suggesting that larger ipsilateral pupillary dilation after a luminance decrease cannot be only attributed to the inhibition of the parasympathetic system, but is also mediated by the excitation of the sympathetic system. Together, our results demonstrated a larger ipsilateral pupil response in both the pupillary light and darkness reflex, indicating an asymmetry in ipsilateral and contralateral neural circuitry of the pupillary darkness reflex.
Subject(s)
Anisocoria/physiopathology , Darkness , Pupil/physiology , Reflex, Pupillary/physiology , Adult , Female , Humans , Light , Male , Photic Stimulation , Visual Fields/physiologyABSTRACT
The adaptation of CRISPR/SpCas9 technology to mammalian cell lines is transforming the study of human functional genomics. Pooled libraries of CRISPR guide RNAs (gRNAs) targeting human protein-coding genes and encoded in viral vectors have been used to systematically create gene knockouts in a variety of human cancer and immortalized cell lines, in an effort to identify whether these knockouts cause cellular fitness defects. Previous work has shown that CRISPR screens are more sensitive and specific than pooled-library shRNA screens in similar assays, but currently there exists significant variability across CRISPR library designs and experimental protocols. In this study, we reanalyze 17 genome-scale knockout screens in human cell lines from three research groups, using three different genome-scale gRNA libraries. Using the Bayesian Analysis of Gene Essentiality algorithm to identify essential genes, we refine and expand our previously defined set of human core essential genes from 360 to 684 genes. We use this expanded set of reference core essential genes, CEG2, plus empirical data from six CRISPR knockout screens to guide the design of a sequence-optimized gRNA library, the Toronto KnockOut version 3.0 (TKOv3) library. We then demonstrate the high effectiveness of the library relative to reference sets of essential and nonessential genes, as well as other screens using similar approaches. The optimized TKOv3 library, combined with the CEG2 reference set, provide an efficient, highly optimized platform for performing and assessing gene knockout screens in human cell lines.
Subject(s)
CRISPR-Cas Systems/genetics , Gene Knockout Techniques , Genetic Testing , Genome , Gene Library , Genes, Essential , HEK293 Cells , Humans , RNA, Guide, Kinetoplastida/genetics , Reference StandardsABSTRACT
The E3 ubiquitin ligase Mule is often overexpressed in human colorectal cancers, but its role in gut tumorigenesis is unknown. Here, we show in vivo that Mule controls murine intestinal stem and progenitor cell proliferation by modulating Wnt signaling via c-Myc. Mule also regulates protein levels of the receptor tyrosine kinase EphB3 by targeting it for proteasomal and lysosomal degradation. In the intestine, EphB/ephrinB interactions position cells along the crypt-villus axis and compartmentalize incipient colorectal tumors. Our study thus unveils an important new avenue by which Mule acts as an intestinal tumor suppressor by regulation of the intestinal stem cell niche.
