Generating and testing the efficacy of reagents for CRISPR/Cas9 homology directed repair-based manipulations in Tribolium.

CRISPR/Cas9 manipulations are possible in many insects and ever expanding. Nonetheless, success in one species and techniques developed for it are not necessarily applicable to other species. As such, the development and expansion of CRISPR-based (clustered regularly interspaced short palindromic repeats) genome-editing tools and methodologies are dependent upon direct experimentation. One useful technique is Cas9-dependent homologous recombination, which is a critical tool for studying gene function but also for developing pest related applications like gene drive. Here, we report our attempts to induce Cas9 homology directed repair (HDR) and subsequent gene drive in Tribolium castaneum (Herbst; Insecta: Coleoptera: Tenebrionidae). Utilizing constructs containing 1 or 2 target gRNAs in combination with Cas9 under 2 different promoters and corresponding homology arms, we found a high incidence of CRISPR/Cas9 induced mutations but no evidence of homologous recombination. Even though the generated constructs provide new resources for CRISPR/Cas9 modification of the Tribolium genome, our results suggest that additional modifications and increased sample sizes will be necessary to increase the potential and detection for HDR of the Tribolium genome.

Inter-transducer variability of ultrasound image quality in obese adults: Qualitative and quantitative comparisons.

PURPOSE: Acquiring high-quality ultrasound images of deep abdominal organs and vasculatures in obese adults (BMI >30 kg/cm2) is considered challenging. The aim of the study was to assess the inter-transducer variability in B-mode and color Doppler image quality from four commercial ultrasound transducers through qualitative and quantitative analyses. METHODS: Four curvilinear transducers on three ultrasound scanners were used to acquire B-mode and color Doppler images of deep abdominal structures in 15 obesity ≥ class II (BMI >35 kg/cm2) adults. Using visual-qualitative assessment and an offline image processing software, visual-qualitative score and quantitative mean pixel values of B-mode images, and color area ratios of color Doppler images were calculated. Differences in these values among the transducers were analyzed using one-way ANOVA. The intra- and inter-observer reliability of visual-qualitative assessment and offline image processing was tested using the intraclass correlation coefficient (ICC). RESULTS: Differences in visual-qualitative score, mean pixel value of B-mode images, and color area ratio of color Doppler images among the four transducers were significant (p < 0.001). Transducer -4 produced the highest quality of B-mode (45-53% improvement) and color Doppler (22-73% improvement) images among the transducers. Intra-observer repeatability and inter-observer reproducibility were higher with performing offline image processing than visual-qualitative assessment (ICC: 0.97-0.99 versus ICC: 0.76-0.97). CONCLUSION: There was significant image quality variability between different transducers. Transducer -4, a transducer designed specifically for high BMI patients, had the highest quality B-mode and color Doppler images compared to the other transducers lending to improved ultrasonographic visualization in obese patients.

A TRiP RNAi screen to identify molecules necessary for Drosophila photoreceptor differentiation.

Drosophila rhabdomeric terminal photoreceptor differentiation is an extended process taking several days to complete. Following ommatidial patterning by the morphogenetic furrow, photoreceptors are sequentially recruited and specified, and terminal differentiation begins. Key events of terminal differentiation include the establishment of apical and basolateral domains, rhabdomere and stalk formation, inter-rhabdomeral space formation, and expression of phototransduction machinery. While many key regulators of these processes have been identified, the complete network of transcription factors to downstream effector molecules necessary for regulating each of these major events remains incomplete. Here, we report an RNAi screen to identify additional molecules and cellular pathways required for photoreceptor terminal differentiation. First, we tested several eye-specific GAL4 drivers for correct spatial and temporal specificity and identified Pph13-GAL4 as the most appropriate GAL4 line for our screen. We screened lines available through the Transgenic RNAi Project and isolated lines that when combined with Pph13-GAL4 resulted in the loss of the deep pseudopupil, as a readout for abnormal differentiation. In the end, we screened 6,189 lines, representing 3,971 genes, and have identified 64 genes, illuminating potential new regulatory molecules and cellular pathways for the differentiation and organization of Drosophila rhabdomeric photoreceptors.

Reliability of performing ultrasound derived SWE and fat fraction in adult livers.

PURPOSE: The aim of the study was to test the reproducibility of performing conventional point shear wave elastography (pSWE), auto-pSWE, and ultrasound derived fat fraction (UDFF) in adult livers. METHODS: The Institutional Review Board approved the study and all participants provided written informed consent. Conventional pSWE (obtaining 10 measurements through 10 acquisitions), auto-pSWE (automatically obtaining 15 measurements by a single acquisition), and UDFF (one measurement obtained by one acquisition) of the liver were prospectively performed in 21 participants (10 men, 11 women, mean age 63y) by senior and junior operators in February-May 2021. Shear wave velocity (SWV, m/s) of the liver was measured by conventional pSWE and auto-pSWE. Intraclass correlation coefficient (ICC) and Bland-Altman limits of agreement were used to test intra-observer repeatability and inter-observer reproducibility in measuring pSWE, auto-pSWE, and UDFF. RESULTS: ICC for testing intra-observer repeatability and inter-observer reproducibility in performing pSWE, auto-pSWE, and UDFF was >0.85 (95% confidence interval 0.85-0.99). The mean difference of 95% Bland-Altman limits of agreement was -0.02 (upper 0.09, lower -0.12) and the correlation of SWV measured between conventional pSWE and auto-pSWE methods was strong (r2 = 0.87). CONCLUSION: Our results suggest good repeatability and reproducibility in measuring UDFF and SWV in adult livers. The auto-pSWE has higher reliability, reproducibility and time efficacy in measuring SWV of adult livers when compared to conventional pSWE method.