A Scalable 3D High-Content Imaging Protocol for Measuring a Drug Induced DNA Damage Response Using Immunofluorescent Subnuclear γH2AX Spots in Patient Derived Ovarian Cancer Organoids.

The high morbidity rate of ovarian cancer has remained unchanged during the past four decades, partly due to a lack of understanding of disease mechanisms and difficulties in developing new targeted therapies. Defective DNA damage detection and repair is one of the hallmarks of cancer cells and is a defining characteristic of ovarian cancer. Most in vitro studies to date involve viability measurements at scale using relevant cancer cell lines; however, the translation to the clinic is often lacking. The use of patient derived organoids is closing that translational gap, yet the 3D nature of organoid cultures presents challenges for assay measurements beyond viability measurements. In particular, high-content imaging has the potential for screening at scale, providing a better understanding of the mechanism of action of drugs or genetic perturbagens. In this study we report a semiautomated and scalable immunofluorescence imaging assay utilizing the development of a 384-well plate based subnuclear staining and clearing protocol and optimization of 3D confocal image analysis for studying DNA damage dose response in human ovarian cancer organoids. The assay was validated in four organoid models and demonstrated a predictable response to etoposide drug treatment with the lowest efficacy observed in the clinically most resistant model. This imaging and analysis method can be applied to other 3D organoid and spheroid models for use in high content screening.

Evaluation of the relationship between para-aortic adipose tissue and ascending aortic diameter using a new method.

BACKGROUND: Para-aortic adipose tissue (PAT) is the local adipose tissue that externally surrounds the aorta. It contributes significantly to aortic atherosclerosis and enlargement. Studies conducted with computed tomography and magnetic resonance have shown that individuals with aortic aneurysm had more PAT than healthy individuals. In this study, we measured PAT using transthoracic echocardiography (TTE). The aim of this study is to investigate the possible relationship of TTE measured PAT with ascending aortic width. METHODS: PAT was defined as the hypoechoic space in front of ascending aortic 2 cm above the sinotubular junction at the end of the systole. Patients were divided into 2 groups according to the presence of dilatation in the ascending aorta using Roman's classification (aortic size index, ASI). ASI of less than 21 was considered no aortic dilation and an ASI of 21 mm/m2 or greater was considered to have aortic dilation. RESULTS: A total of 321 unselected patients were divided into the ascending aortic dilatation (AAD) group (n = 96) and the normal ascending aorta diameter group (n = 225 patients). PAT was significantly higher in the AAD group compared with the non-ADD group (0.9 (0.48) vs. 0.7 (0.91) mm, p < 0.0001). Univariate and multivariate logistic regression analysis revealed that PAT (OR: 3.005, 95%CI (1.445-6.251)) were significantly associated with AAD. CONCLUSIONS: This is the first study which evaluated PAT measured by TTE. We found a significant association between PAT measured by TTE and ascending aorta width.

Human rhinovirus promotes STING trafficking to replication organelles to promote viral replication.

Human rhinovirus (HRV), like coronavirus (HCoV), are positive-strand RNA viruses that cause both upper and lower respiratory tract illness, with their replication facilitated by concentrating RNA-synthesizing machinery in intracellular compartments made of modified host membranes, referred to as replication organelles (ROs). Here we report a non-canonical, essential function for stimulator of interferon genes (STING) during HRV infections. While the canonical function of STING is to detect cytosolic DNA and activate inflammatory responses, HRV infection triggers the release of STIM1-bound STING in the ER by lowering Ca2+, thereby allowing STING to interact with phosphatidylinositol 4-phosphate (PI4P) and traffic to ROs to facilitates viral replication and transmission via autophagy. Our results thus hint a critical function of STING in HRV viral replication and transmission, with possible implications for other RO-mediated RNA viruses.

Relationship between Aortic Arch Calcification, Detected by Chest X-Ray, and Renal Resistive Index in Patients with Hypertension.

OBJECTIVE: Aortic arch calcification (AAC) is a surrogate marker for arterial stiffness and hypertension-related vascular damage. Renal resistive index (RRI), a renal Doppler ultrasonography parameter, is used to assess renal hemodynamics. In this study, we aimed to evaluate the relationship between RRI and AAC in patients with hypertension. METHODS: Patients with hypertension underwent a chest X-ray and re nal Doppler ultrasonography. They were divided into two groups according to RRI (group 1: RRI ≥0.70; group 2: RRI < 0.70). Two examiners, blinded to the findings of RRI, reviewed the AAC in these patients. The kappa value was detected to be 0.781 and a p value < 0.001 was considered significant. RESULTS: The study included 289 hypertensive patients (mean age 63.87 ± 11.38 years). In 53.6% (n = 155) of the study subjects, RRI was observed to be ≥0.70. Patients with RRI ≥70 were older and had more prevalent AAC as well as left ventricular hypertrophy. A multiple linear regression analysis was carried out to test whether presence of AAC significantly predicted RRI. The results of the regression analysis indicated that presence of AAC significantly predicted RRI (ß = 0.053; p < 0.001). CONCLUSIONS: A strong and independent relationship was found between AAC on chest X-ray and RRI in patients with hypertension.

Characterization of hawthorn (Crataegus spp.) genotypes by SSR markers.

Hawthorn (Crataegus spp.) is an edible wild fruit that is used in traditional medicine, landscape studies, and food and beverage industries in many countries. It is an important wild plant species in Turkey and is numerous in the Yozgat Province. Genetic and breeding studies on hawthorn are very limited. Therefore, we aimed to characterize 91 hawthorn genotypes using simple sequence repeat (SSR) markers. The SSRs were developed from apple and pear and were screened in hawthorn for amplification and polymorphisms. A total of 265 alleles were detected from thirty-two SSR primer pairs, and those were used to identify genetic relationships. The number of alleles ranged from 2 to 21 alleles per locus with a mean value of 8.28. The Hi05b09 locus showed the highest allele number (Na = 21). The polymorphism information content (PIC) values ranged from 0.16 (CH03d10) to 0.89 (C6554) with a mean value of 0.60. An Unweighted Pair Group Method with Arithmetic Average method was used to cluster the genotypes, and four major clusters were obtained from the amplification of the SSRs. STRUCTURE software identified four populations (ΔK = 4) and eight sub-populations (ΔK = 8), and four major clusters similar results to UPGMA analysis. Our study showed that the SSR markers could be utilized as a reliable tool for the determination of genetic variations and relationships of hawthorn genotypes. A basic molecular analysis on the hawthorn genotypes identified in this study will promote the collection of germplasm collection and the selection of parents' in future cross-breeding studies.

Onychomycosis is associated with subclinical atherosclerosis in patients with diabetes.

BACKGROUND: We aimed to investigate the association of toenail onychomycosis with subclinical atherosclerosis in patients with diabetes mellitus. PATIENTS AND METHODS: Consecutive diabetic patients who were seen at our outpatient clinic were enrolled. The carotid intima-media thickness (CIMT) was assessed and toenail onychomycosis was diagnosed with microscopic evaluation. RESULTS: We investigated 127 patients with diabetes melltus type 2. Overall, the prevalence of toenail onychomycosis was 37.8 % (48 of 127). Of the 127 patients, 60 (47.2 %) had subclinical atherosclerosis (CIMT ≥ 1 mm). Prevalence of male gender (43.3 % vs. 22.4 %, p = 0.012) and onychomycosis (53.3 % vs. 23.9 %, p = 0.001) was significantly higher in patients with subclinical atherosclerosis. Among biochemical parameters, low-density lipoprotein (122 ± 38 mg/dL vs. 108 ± 36 mg/dL, p = 0.039) and glycosylated hemoglobin levels (median 8.4 %, IQR: 2.1 % vs. median 7.5 %, IQR: 1.6 %, p = 0.002) were significantly higher in patients with subclinical atherosclerosis. Study groups were similar with respect to all other demographic, clinical, and laboratory parameters. After adjustment for all potential confounders, the presence of onychomycosis was independently associated with subclinical atherosclerosis (OR 2.77, 95 % CI 1.16 to 6.30) in multivariate logistic regression analysis. CONCLUSIONS: Presence of onychomycosis in patients with diabetes is associated with subclinical atherosclerosis. Onychomycosis may be a marker of atherosclerotic arterial involvement.

The application of non-linear curve fitting routines to the analysis of mid-infrared images obtained from single polymeric microparticles.

For the first time, we report a series of time resolved images of a single PLGA microparticle undergoing hydrolysis at 70 °C that have been obtained using attenuated total reflectance-Fourier transform infrared spectroscopic (ATR-FTIR) imaging. A novel partially supervised non-linear curve fitting (NLCF) tool was developed to identify and fit peaks to the infrared spectrum obtained from each pixel within the 64 × 64 array. The output from the NLCF was evaluated by comparison with a traditional peak height (PH) data analysis approach and multivariate curve resolution alternating least squares (MCR-ALS) analysis for the same images, in order to understand the limitations and advantages of the NLCF methodology. The NLCF method was shown to facilitate consistent spatial resolution enhancement as defined using the step-edge approach on dry microparticle images when compared to images derived from both PH measurements and MCR-ALS. The NLCF method was shown to improve both the S/N and sharpness of images obtained during an evolving experiment, providing a better insight into the magnitude of hydration layers and particle dimension changes during hydrolysis. The NLCF approach facilitated the calculation of hydrolysis rate constants for both the glycolic (kG) and lactic (kL) acid segments of the PLGA copolymer. This represents a real advantage over MCR-ALS which could not distinguish between the two segments due to colinearity within the data. The NLCF approach made it possible to calculate the hydrolysis rate constants from a single pixel, unlike the peak height data analysis approach which suffered from poor S/N at each pixel. These findings show the potential value of applying NLCF to the study of real-time chemical processes at the micron scale, assisting in the understanding of the mechanisms of chemical processes that occur within microparticles and enhancing the value of the mid-IR ATR analysis.