Interactive youth science workshops benefit student participants and graduate student mentors.

Science communication and outreach are essential for training the next generation of scientists and raising public awareness for science. Providing effective science, technology, engineering, and mathematics (STEM) educational outreach to students in classrooms is challenging because of the need to form partnerships with teachers, the time commitment required for the presenting scientist, and the limited class time allotted for presentations. In our Present Your Ph.D. Thesis to a 12-Year Old outreach project, our novel solution to this problem is hosting a youth science workshop (YSW) on our university campus. The YSW is an interpersonal science communication and outreach experience in which graduate students from diverse scientific disciplines introduce middle and high school students to their cutting-edge research and mentor them to develop a white-board presentation to communicate the research to the workshop audience. Our assessment of the YSW indicated that participating young students expressed significantly more positive attitudes toward science and increased motivation to work in a STEM career after attending the workshop. Qualitative follow-up interviews with participating graduate students' show that even with minimal time commitment, an impactful science communication training experience can be achieved. The YSW is a low-cost, high-reward educational outreach event amenable to all disciplines of science. It enhances interest and support of basic science research while providing opportunities for graduate students to engage with the public, improve their science communication skills, and enhance public understanding of science. This YSW model can be easily implemented at other higher education institutions to globally enhance science outreach initiatives.

Identification of lipid biomarkers of metastatic potential and gene expression (HER2/p53) in human breast cancer cell cultures using ambient mass spectrometry.

In breast cancer, overexpression of human epidermal growth factor receptor 2 (HER2) correlates with overactivation of lipogenesis, mutation of tumor suppressor p53, and increased metastatic potential. The mechanisms through which lipids mediate p53, HER2, and metastatic potential are largely unknown. We have developed a desorption electrospray ionization mass spectrometry (DESI-MS) method to identify lipid biomarkers of HER2/p53 expression, metastatic potential, and disease state (viz. cancer vs. non-cancerous) in monolayer and suspension breast cancer cell cultures (metastatic potential: MCF-7, T-47D, MDA-MB-231; HER2/p53: HCC2218 (HER2+++/p53+), HCC1599 (HER2-/p53-), HCC202 (HER2++/p53-), HCC1419 (HER2+++/p53-) HCC70 (HER2-/p53+++); non-cancerous: MCF-10A). Unsupervised principal component analysis (PCA) of DESI-MS spectra enabled identification of twelve lipid biomarkers of metastatic potential and disease state, as well as ten lipids that distinguish cell lines based on HER2/p53 expression levels (> 200 lipids were identified per cell line). In addition, we developed a DESI-MS imaging (DESI-MSI) method for mapping the spatial distribution of lipids in metastatic spheroids (MDA-MB-231). Of the twelve lipids that correlate with changes in the metastatic potential of monolayer cell cultures, three were localized to the necrotic core of spheroids, indicating a potential role in promoting cancer cell survival in nutrient-deficient environments. One lipid species, which was not detected in monolayer MDA-MB-231 cultures, was spatially localized to the periphery of the spheroid, suggesting a potential role in invasion and/or proliferation. These results demonstrate that combining DESI-MS/PCA of monolayer and suspension cell cultures with DESI-MSI of spheroids is a promising approach for identifying lipid biomarkers of specific genotypes and phenotypes, as well as elucidating the potential function of these biomarkers in breast cancer. Graphical Absract.

Synchronized Desorption Electrospray Ionization Mass Spectrometry Imaging.

Desorption electrospray ionization (DESI) has emerged as a powerful technique for mass spectral analysis and imaging under ambient conditions. Synchronization of DESI (sDESI) with the ion injection period (IT)of low-duty cycle mass spectrometers has been previously shown to improve sensitivity and reduce the amount of sample depleted during the acquisition of each spectrum (viz. MS scan time). In this report, we describe the development and characterization of an sDESI mass spectrometry imaging source (sDESI-MSI). Our results show that synchronization of DESI with the IT of an LTQ Orbitrap-XL mass spectrometer improves spatial resolution by factors of ∼4-6. In addition, under certain experimental conditions, synchronization was essential to acquire distinct MS images of low-intensity endogenous FAs (< 5% relative intensity) in fingermarks at high sampling frequencies (step sizes ≤ 75 µm). The magnitudes of these improvements in performance depend on the properties of the microdroplet spray, sample, and surface. Simulations that model analyte movement during desorption and the "washing effect" replicate the experimental results with the washing parameter having the greatest impact on performance. Thus, synchronization improves spatial resolution and sensitivity by decreasing the percentage of the total MS scan time that analytes are influenced by the "washing effect". Generally, synchronization of DESI with IT improves performance and expands the range of analytes, surfaces, and experimental conditions amenable to DESI-MSI, especially for analytes that are weakly attached to a surface.

Identification of a complex intrachromosomal inverted insertion in the long arm of chromosome 9 as a cause of tuberous sclerosis complex in a Korean family.

BACKGROUND: Tuberous sclerosis complex (TSC) is an autosomal dominant multisystem disorder, caused by a loss-of-function of either TSC1 or TSC2 gene. However, in 10%-15% TSC patients there is no pathogenic variant identified in either TSC1 or TSC2 genes based on standard clinical testing. METHODS: In this study, genome sequencing was performed for families with clinical diagnosis of TSC with negative results from TSC1 and TSC2 single-gene tests. RESULTS: Herein, we report a family presenting a classical TSC phenotype with an unusual, complex structural variant involving the TSC1 gene: an intrachromosomal inverted insertion in the long arm of chromosome 9. We speculate that the inverted 9q33.3q34.13 region was inserted into the q31.2 region with the 3'-end of the breakpoint of the inversion being located within the TSC1 gene, resulting in premature termination of TSC1. CONCLUSIONS: In this study, we demonstrate the utility of genome sequencing for the identification of complex chromosomal rearrangement. Because the breakpoints are located within the deep intronic/intergenic region, this copy-neutral variant was missed by the TSC1 and TSC2 single-gene tests and contributed to an unknown etiology. Together, this finding suggests that complex structural variants may be underestimated causes for the etiology of TSC.

Cross-Sectional Analysis of Exome Sequencing Diagnosis in Patients With Neurologic Phenotypes Facing Barriers to Clinical Testing.

Background and Objectives: Exome sequencing (ES) demonstrates a 20-50 percent diagnostic yield for patients with a suspected monogenic neurologic disease. Despite the proven efficacy in achieving a diagnosis for such patients, multiple barriers for obtaining exome sequencing remain. This study set out to assess the efficacy of ES in patients with primary neurologic phenotypes who were appropriate candidates for testing but had been unable to pursue clinical testing. Methods: A total of 297 patients were identified from the UCLA Clinical Neurogenomics Research Center Biobank, and ES was performed, including bioinformatic assessment of copy number variation and repeat expansions. Information regarding demographics, clinical indication for ES, and reason for not pursuing ES clinically were recorded. To assess diagnostic efficacy, variants were interpreted by a multidisciplinary team of clinicians, bioinformaticians, and genetic counselors in accordance with the American College of Medical Genetics and Genomics variant classification guidelines. We next examined the specific barriers to testing for these patients, including how frequently insurance-related barriers such as coverage denials and inadequate coverage of cost were obstacles to pursuing exome sequencing. Results: The cohort primarily consisted of patients with sporadic conditions (n = 126, 42.4%) of adult-onset (n = 239, 80.5%). Cerebellar ataxia (n = 225, 75.8%) was the most common presenting neurologic phenotype. Our study found that in this population of mostly adult patients with primary neurologic phenotypes that were unable to pursue exome sequencing clinically, 47 (15.8%) had diagnostic results while an additional 24 patients (8.1%) had uncertain results. Of the 297 patients, 206 were initially recommended for clinical exome but 88 (42.7%) could not pursue ES because of insurance barriers, of whom 14 (15.9%) had diagnostic findings, representing 29.8% of all patients with diagnostic findings. In addition, the incorporation of bioinformatic repeat expansion testing was valuable, identifying a total of 8 pathogenic repeat expansions (17.0% of all diagnostic findings) including 3 of the common spinocerebellar ataxias and 2 patients with Huntington disease. Discussion: These findings underscore the importance and value of clinical ES as a diagnostic tool for neurogenetic disease and highlight key barriers that prevent patients from receiving important clinical information with potential treatment and psychosocial implications for patients and family members.

Unveiling genetics of non-syndromic albinism using whole exome sequencing: A comprehensive study of TYR, TYRP1, OCA2 and MC1R genes in 17 families.

BACKGROUND: Oculocutaneous albinism (OCA) is a group of skin depigmentation disorders. Clinical presentation of OCA includes defects in melanocyte differentiation, melanin biosynthesis, and melanosome maturation and transport. OBJECTIVES: A molecular diagnostics study of families presenting oculocutaneous albinism. METHODS: In this study, 17 consanguineous OCA families consisting of 93 patients were investigated. Whole Exome Sequencing (WES) of the index patient in each family were performed. Short listed variants of WES were Sanger validated for Mendelian segregation in obligate carriers and other available family members. Variant prioritization and pathogenicity were classified as per the criteria of American College Medical Genetics and Genomics (ACMG). Comparative computational modelling was performed to predict the potential damaging effect of the altered proteins. RESULTS: 15 pathogenic variations: c.132 T > A, c.346C > T, c.488C > G, c.1037G > A in TYR, c.1211C > T, c.1441G > A, c.1706_1707insT, c.2020C > G, c.2402G > C, c.2430del, in OCA2, c.1067G > A in TYRP1 and c.451C > T, c.515G > T, c.766C > T, c.917G > A in MC1R genes were identified. Three variants in OCA2 gene were characterized: c.1706_1707insT, c.2430del, and c.2402G > C, all of which were not reported before in OCA families. CONCLUSION: A few studies focusing on mutation screening of OCA patients have been reported before; however, this study has uniquely presents the Pakhtun ethnic population residing on the North-Western boarder. It explains that TYR, OCA2, TYRP1, and MC1R variations lead to non-syndromic OCA phenotype The overlapping phenotypes of OCA can precisely be diagnosed for its molecular pathogenicity using WES. This study recommends WES as a first-line molecular diagnostic tool, and provides a basis for developing customized genetic tests i.e. pre-marital screening to reduce the disease burden in the future generations.

Whole exome sequencing identified five novel variants in CNTN2, CARS2, ARSA, and CLCN4 leading to epilepsy in consanguineous families.

Introduction: Epilepsy is a group of neurological disorders characterized by recurring seizures and fits. The Epilepsy genes can be classified into four distinct groups, based on involvement of these genes in different pathways leading to Epilepsy as a phenotype. Genetically the disease has been associated with various pathways, leading to pure epilepsy-related disorders caused by CNTN2 variations, or involving physical or systemic issues along with epilepsy caused by CARS2 and ARSA, or developed by genes that are putatively involved in epilepsy lead by CLCN4 variations. Methods: In this study, five families of Pakistani origin (EP-01, EP-02, EP-04, EP-09, and EP-11) were included for molecular diagnosis. Results: Clinical presentations of these patients included neurological symptoms such as delayed development, seizures, regression, myoclonic epilepsy, progressive spastic tetraparesis, vision and hearing impairment, speech problems, muscle fibrillation, tremors, and cognitive decline. Whole exome sequencing in index patients and Sanger sequencing in all available individuals in each family identified four novel homozygous variants in genes CARS2: c.655G>A p.Ala219Thr (EP-01), ARSA: c.338T>C: p.Leu113Pro (EP-02), c.938G>T p.Arg313Leu (EP-11), CNTN2: c.1699G>T p.Glu567Ter (EP-04), and one novel hemizygous variant in gene CLCN4: c.2167C>T p.Arg723Trp (EP-09). Conclusion: To the best of our knowledge these variants were novel and had not been reported in familial epilepsy. These variants were absent in 200 ethnically matched healthy control chromosomes. Three dimensional protein analyses revealed drastic changes in the normal functions of the variant proteins. Furthermore, these variants were designated as "pathogenic" as per guidelines of American College of Medical Genetics 2015. Due to overlapping phenotypes, among the patients, clinical subtyping was not possible. However, whole exome sequencing successfully pinpointed the molecular diagnosis which could be helpful for better management of these patients. Therefore, we recommend that exome sequencing be performed as a first-line molecular diagnostic test in familial cases.

A comparative histological and immunohistochemical study of wound healing following incision with a scalpel, CO2 laser or Er,Cr:YSGG laser in the guinea pig oral mucosa.

PURPOSE: This study was undertaken to compare wound healing following incisions with either a scalpel, CO(2) laser or Er,Cr:YSGG laser in Guinea pig oral mucosa. MATERIALS AND METHODS: Three types of wounds were randomly introduced with either a stainless steel scalpel, CO(2) laser or Er,Cr:YSGG laser in the buccal mucosa of each of 22 Guinea pigs. Four Guinea pigs were sacrificed on day 1, day 3 and day 5 post-surgery. Five Guinea pigs were sacrificed on day 7 and day 14 post-surgery. Biopsy samples from each oral mucosa wound were examined and the expression of TNF-α and TGF-ß1 was determined by immunohistochemical staining. RESULTS: At day 3 post-surgery, the histological pattern of the healing process was similar in the scalpel and Er,Cr:YSGG laser wounds and there were more ulcerations present in the CO(2) laser wounds than in the scalpel and Er,Cr:YSGG laser wounds. The level of TNF-α expression was twice in the laser wounds that in the scalpel wounds. A higher level of TGF-ß1 expression was seen at day 7 post-surgery and a lower level at day 14 post-surgery in the CO(2) laser wounds than in the scalpel and Er,Cr:YSGG laser wounds. CONCLUSIONS: The Er,Cr:YSGG laser has many advantages for oral surgery due to a low inflammatory response and minimal damage of the tissue. Although a CO(2) laser has better hemostatic ability, its use causes greater tissue damage than a scalpel and Er,Cr:YSGG laser. However, further larger studies would be needed before fully endorsing its widespread use.

Surgical Treatment for Left Main Coronary Atresia with Significant Mitral Regurgitation in a 1-Year-Old Child.

Congenital atresia of the left main coronary artery (LMCA) is an extremely rare coronary anomaly that necessitates surgical correction. Patients with LMCA atresia may have various clinical symptoms, which are determined by the degree of collateral vessel development from the right coronary system, the metabolic demands of the heart, and concomitant mitral insufficiency caused by myocardial ischemia. Unlike in adults, there are limited surgical options for coronary artery disease in children. Herein, we report a case of LMCA atresia with mitral regurgitation in a 19-month-old child that was successfully corrected by coronary artery bypass grafting and mitral valve repair.

Surgical Outcomes of Malignant Primary Cardiac Tumor: A 20-Year Study at a Single Center.

BACKGROUND: Malignant primary cardiac tumors are extremely rare, but have a poor prognosis. This study evaluated the surgical outcomes of patients with this disease. METHODS: Forty patients who underwent surgery for malignant primary cardiac tumors between January 1998 and December 2018 were enrolled. Participants were divided into 3 groups based on resection margins (R0, 14 patients; R1, 11 patients; and R2, 11 patients) and their surgical outcomes were compared. Heart transplantation was performed in 4 patients with unresectable tumors. RESULTS: Early mortality was reported in 2 cases (5%) due to postoperative bleeding and cerebral hemorrhage secondary to brain metastasis. The 1- and 2-year survival rates were 67.5% and 42.5%, respectively. The median survival time of the patients was 20.3 months (range, 9.2-37.6 months). The median survival time was 48.7, 20.3, and 4.8 months in patients with R0, R1, and R2 resections, respectively (p=0.023). Tumor recurrence occurred in 21 patients (61.7%), including 4 cases of local recurrence and 17 cases of distant metastasis. In patients who underwent heart transplantation, the median survival time was 29.5 months, with 3 cases of distant metastasis. CONCLUSION: Although surgery for malignant primary cardiac tumors has a poor prognosis, complete resection of the tumor may improve surgical outcomes.

Cardiolipins Are Biomarkers of Mitochondria-Rich Thyroid Oncocytic Tumors.

Oncocytic tumors are characterized by an excessive eosinophilic, granular cytoplasm due to aberrant accumulation of mitochondria. Mutations in mitochondrial DNA occur in oncocytic thyroid tumors, but there is no information about their lipid composition, which might reveal candidate theranostic molecules. Here, we used desorption electrospray ionization mass spectrometry (DESI-MS) to image and chemically characterize the lipid composition of oncocytic thyroid tumors, as compared with nononcocytic thyroid tumors and normal thyroid samples. We identified a novel molecular signature of oncocytic tumors characterized by an abnormally high abundance and chemical diversity of cardiolipins (CL), including many oxidized species. DESI-MS imaging and IHC experiments confirmed that the spatial distribution of CLs overlapped with regions of accumulation of mitochondria-rich oncocytic cells. Fluorescent imaging and mitochondrial isolation showed that both mitochondrial accumulation and alteration in CL composition of mitochondria occurred in oncocytic tumors cells, thus contributing the aberrant molecular signatures detected. A total of 219 molecular ions, including CLs, other glycerophospholipids, fatty acids, and metabolites, were found at increased or decreased abundance in oncocytic, nononcocytic, or normal thyroid tissues. Our findings suggest new candidate targets for clinical and therapeutic use against oncocytic tumors. Cancer Res; 76(22); 6588-97. ©2016 AACR.