Screen for Photoreceptor and Other Eyespot Defective Mutants in Chlamydomonas

Chlamydomonas are single celled photosynthetic organisms that rely on their single eyespot to help them navigate towards light for use as an energy source. Their eyespot is crucial for their survival and as a result, the genetics behind the formation of the eyespot is regularly studied. To study and analyze several specific genes thought to be involved in eyespot function and formation, I collected phototaxis-defective mutants after mutagenic transformation of the arg7-2 auxotrophic strain with the ARG7 gene (arginine biosynthesis). Transformation of the Chlamydomonas genome was done by electroporation. The genes targeted in this study code for COP4 (photoreceptor), SOUL3 (affects eyespot size and placement), the MORN Repeat (attaches proteins to the membrane), MEC17 and MAP65 (affects tubulin dynamics), and the (PAP)-fibrillin domain (stabilizes pigment granule arrays) proteins. The ARG7 gene inserted into the Chlamydomonas genome randomly, and I intended to screen ultimately for strains with specific disruptions in these genes. The electroporation transformants were collected and analyzed using phototaxis techniques in order to find strains containing mutations that affect the functionality of the eyespot. Phototaxis-deficient mutants will be screened by PCR to identify those with disruptions in any of the targeted genes. Fluorescence and light microscopy techniques will be used to identify the mutant phenotypes and sequencing will determine the precise location of the gene insertion

Investigation of the dynamic properties of on-chip coupled piezo/photodiodes by time-resolved atomic force and Kelvin probe microscopy

We have studied the dynamic properties of hybrid devices in which the piezoelectric material lead zirconate titanate is integrated with silicon photodiodes on-chip. Such an integrated system enables direct conversion of light energy into mechanical deformation and motion, opening up new pathways for light propulsion in microrobots and nanorobots. By operating our devices under alternating illumination and simultaneously recording the time-dependent deformation and surface potential, we were able to derive frequency and voltage dependent time constants and phase relations between photovoltage and deformation. We observed that the silicon top contact resistance limits the response time to 6 ms in small area devices in which the capacitance is low. Furthermore, we observed a phase transition at low frequency that seems to be consistent with the occurrence of a negative capacitance. Our method of using time-dependent atomic force and Kelvin probe force microscopy proves to be suitable for the investigation of nanoscale, dynamic properties of light-driven piezo systems and can lead the design of next generation devices

Functional repair assay for the diagnosis of constitutional mismatch repair deficiency from non-neoplastic tissue

Purpose: Constitutional mismatch repair deficiency (CMMRD) is a highly penetrant cancer predisposition syndrome caused by biallelic mutations in mismatch repair (MMR) genes. As several cancer syndromes are clinically similar, accurate diagnosis is critical to cancer screening and treatment. As genetic diagnosis is confounded by 15 or more pseudogenes and variants of uncertain significance, a robust diagnostic assay is urgently needed. We sought to determine whether an assay that directly measures MMR activity could accurately diagnose CMMRD.Patients and Methods: In vitro MMR activity was quantified using a 3\u27-nicked G-T mismatched DNA substrate, which requires MSH2-MSH6 and MLH1-PMS2 for repair. We quantified MMR activity from 20 Epstein-Barr virus-transformed lymphoblastoid cell lines from patients with confirmed CMMRD. We also tested 20 lymphoblastoid cell lines from patients who were suspected for CMMRD. We also characterized MMR activity from patients with neurofibromatosis type 1, Li-Fraumeni syndrome, polymerase proofreading-associated cancer syndrome, and Lynch syndrome.Results: All CMMRD cell lines had low MMR activity (n = 20; mean, 4.14 ± 1.56%) relative to controls (n = 6; mean, 44.00 ± 8.65%; P \u3c .001). Repair was restored by complementation with the missing protein, which confirmed MMR deficiency. All cases of patients with suspected CMMRD were accurately diagnosed. Individuals with Lynch syndrome (n = 28), neurofibromatosis type 1 (n = 5), Li-Fraumeni syndrome (n = 5), and polymerase proofreading-associated cancer syndrome (n = 3) had MMR activity that was comparable to controls. To accelerate testing, we measured MMR activity directly from fresh lymphocytes, which yielded results in 8 days.Conclusion: On the basis of the current data set, the in vitro G-T repair assay was able to diagnose CMMRD with 100% specificity and sensitivity. Rapid diagnosis before surgery in non-neoplastic tissues could speed proper therapeutic management

Survival Benefit for Individuals With Constitutional Mismatch Repair Deficiency Undergoing Surveillance

Purpose: Constitutional mismatch repair deficiency syndrome (CMMRD) is a lethal cancer predisposition syndrome characterized by early-onset synchronous and metachronous multiorgan tumors. We designed a surveillance protocol for early tumor detection in these individuals. Patients and methods: Data were collected from patients with confirmed CMMRD who were registered in the International Replication Repair Deficiency Consortium. Tumor spectrum, efficacy of the surveillance protocol, and malignant transformation of low-grade lesions were examined for the entire cohort. Survival outcomes were analyzed for patients followed prospectively from the time of surveillance implementation. Results: A total of 193 malignant tumors in 110 patients were identified. Median age of first cancer diagnosis was 9.2 years (range: 1.7-39.5 years). For patients undergoing surveillance, all GI and other solid tumors, and 75% of brain cancers were detected asymptomatically. By contrast, only 16% of hematologic malignancies were detected asymptomatically (P < .001). Eighty-nine patients were followed prospectively and used for survival analysis. Five-year overall survival (OS) was 90% (95% CI, 78.6 to 100) and 50% (95% CI, 39.2 to 63.7) when cancer was detected asymptomatically and symptomatically, respectively (P = .001). Patient outcome measured by adherence to the surveillance protocol revealed 4-year OS of 79% (95% CI, 54.8 to 90.9) for patients undergoing full surveillance, 55% (95% CI, 28.5 to 74.5) for partial surveillance, and 15% (95% CI, 5.2 to 28.8) for those not under surveillance (P < .0001). Of the 64 low-grade tumors detected, the cumulative likelihood of transformation from low-to high-grade was 81% for GI cancers within 8 years and 100% for gliomas in 6 years. Conclusion: Surveillance and early cancer detection are associated with improved OS for individuals with CMMRD