PurposePrimary open angle glaucoma–associated mutations in myocilin (MYOC) cause protein “nonsecretion,” rendering secreted MYOC difficult to detect using conventional techniques. This study focused on developing and using an assay that can quickly and easily detect mutant MYOC secretion.MethodsWe fused Gaussia luciferase (eGLuc2) to MYOC variants and expressed the constructs in HEK-293T and NTM-5 cells. Secreted and intracellular levels of MYOC eGLuc2 variants were evaluated by Western blotting and compared to untagged and FLAG-tagged MYOC constructs. Secreted and soluble intracellular MYOC eGLuc2 were measured by a GLuc assay. The secretion of nine additional MYOC mutants was assayed in conditioned media from transfected cells to test the applicability of the assay for monitoring other MYOC variants.ResultsMyocilin eGLuc2 behaved similarly to untagged and FLAG-tagged MYOC with respect to secretion, soluble intracellular levels, and in response to drug treatment. The GLuc assay could sensitively detect Y437H MYOC secretion 30 minutes after media change. Gaussia luciferase fused variants followed anticipated trends; nonpathogenic variants (D208E, G244V) were secreted at wild-type (WT) levels, whereas predicted disease-causing variants (C245Y, G246R, E300K, Y437H, I477N) demonstrated substantial secretion defects. Secretion defects caused by the C245Y, G246R, and Y437H mutations were partially rescued by permissive growth temperature. Interestingly, however, this increase in secretion was independent of newly synthesized protein.ConclusionsFusion of eGLuc2 to MYOC does not significantly change the behavior of MYOC. This newly developed MYOC reporter system can be used to study engineered MYOC variants and potentially to identify modulators of MYOC secretion and function.

Integrating nephelometers are commonly used to monitor airborne particulate matter. However, they must be calibrated prior to use. The Rayleigh scattering coefficients (b(RS), Mm(-1)), scattering cross sections (σ(RS), cm(2)), and Rayleigh multipliers for tetrafluoromethane (R-14), sulfur hexafluoride, pentafluoroethane (HFC-125), hexafluoropropene (HFC-216), 1,1,1,2,3,3,3,-heptafluoropropane (HFC-227ea), and octafluorocyclobutane (C-318) are reported from measurements made using a Radiance Research M903 integrating nephelometer operating at λ = 530 nm and calibration with gases of known scattering constants. Rayleigh multipliers (±90% conf. int.) were found to be 2.6 ± 0.5, 6.60 ± 0.07, 7.5 ± 1, 14.8 ± 0.9, 15.6 ± 0.5, and 22.3 ± 0.8 times that of air, respectively. To the best of our knowledge, these are the first reported values for R-14, HFC-216, HFC-125, and C-318. Experimental accuracy is supported through measurements of values for SF(6) and HFC-227ea which agree to within 3% of previous literature reports. In addition to documenting fundamental Rayleigh scattering data for the first time, the information presented within will find use for calibration of optical scattering sensors such as integrating nephelometers.

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