Hydrogel Based 3-Dimensional (3D) System for Toxicity and High-Throughput (HTP) Analysis for Cultured Murine Ovarian Follicles

Various toxicants, drugs and their metabolites carry potential ovarian toxicity. Ovarian follicles, the functional unit of the ovary, are susceptible to this type of damage at all stages of their development. However, despite of the large scale of potential negative impacts, assays that study ovarian toxicity are limited. Exposure of cultured ovarian follicles to toxicants of interest served as an important tool for evaluation of toxic effects for decades. Mouse follicles cultured on the bottom of a culture dish continue to serve an important approach for mechanistic studies. In this paper, we demonstrated the usefulness of a hydrogel based 3-dimensional (3D) mouse ovarian follicle culture as a tool to study ovarian toxicity in a different setup. The 3D in vitro culture, based on fibrin alginate interpenetrating network (FA-IPN), preserves the architecture of the ovarian follicle and physiological structure-function relationship. We applied the novel 3D high-throughput (HTP) in vitro ovarian follicle culture system to study the ovotoxic effects of an anti-cancer drug, Doxorobucin (DXR). The fibrin component in the system is degraded by plasmin and appears as a clear circle around the encapsulated follicle. The degradation area of the follicle is strongly correlated with follicle survival and growth. To analyze fibrin degradation in a high throughput manner, we created a custom MATLAB® code that converts brightfield micrographs of follicles encapsulated in FA-IPN to binary images, followed by image analysis. We did not observe any significant difference between manually processed images to the automated MATLAB® method, thereby confirming that the automated program is suitable to measure fibrin degradation to evaluate follicle health. The cultured follicles were treated with DXR at concentrations ranging from 0.005 nM to 200 nM, corresponding to the therapeutic plasma levels of DXR in patients. Follicles treated with DXR demonstrated decreased survival rate in greater DXR concentrations. We observed partial follicle survival of 35% ± 3% (n = 80) in 0.01nM treatment and 48% ± 2% (n = 92) in 0.005nM, which we identified as the IC50 for secondary follicles. In summary, we established a 3D in vitro ovarian follicle culture system that could be used in an HTP approach to measure toxic effects on ovarian follicles.     

Baseline Correlates of Insulin Resistance in Inner City High‐BMI African‐American Children

To characterize the influence of diet‐, physical activity–, and self‐esteem‐related factors on insulin resistance in 8–10‐year‐old African‐American (AA) children with BMI greater than the 85th percentile who were screened to participate in a community‐based type 2 diabetes mellitus (T2DM) prevention trial. In 165 subjects, fasting glucose‐ and insulin‐derived values for homeostasis model assessment of insulin resistance (HOMA‐IR) assessed insulin resistance. Body fatness was calculated following bioelectrical impedance analysis, and fitness was measured using laps from a 20‐m shuttle run. Child questionnaires assessed physical activity, dietary habits, and self‐esteem. Pubertal staging was assessed using serum levels of sex hormones. Parent questionnaires assessed family demographics, family health, and family food and physical activity habits. Girls had significantly higher percent body fat but similar anthropometric measures compared with boys, whereas boys spent more time in high‐intensity activities than girls. Scores for self‐perceived behavior were higher for girls than for boys; and girls desired a more slender body. Girls had significantly higher insulin resistance (HOMA‐IR), compared with boys (P < 0.01). Adjusting for age, sex, pubertal stage, socioeconomic index (SE index), and family history of diabetes, multivariate regression analysis showed that children with higher waist circumference (WC) (P < 0.001) and lower Harter's scholastic competence (SC) scale (P = 0.044) had higher insulin resistance. WC and selected self‐esteem parameters predicted insulin resistance in high‐BMI AA children. The risk of T2DM may be reduced in these children by targeting these factors.     

RNAi and RNAa--the yin and yang of RNAome

RNA interference (RNAi) is a powerful technology with huge applications for functional genomics, target identification in drug discovery and elucidation of molecular signaling pathways. Current RNAi studies have demonstrated the clinical potential of small interfering RNAs (siRNAs) in metabolic diseases, cancer, AIDS, malaria, neurodegenerative disorders, dental diseases and other illnesses. Interestingly, recent studies have shown that the small RNA molecules, either indigenously produced as microRNAs (miRNAs) or exogenously administered synthetic dsRNAs could effectively activate a particular gene in a sequence specific manner instead of silencing it. This novel, but still uncharacterized, phenomenon has been termed as RNA activation (RNAa). The paradoxical concept of Yin and Yang, which describe two primal opposing but complementary principles, can potentially be applied to elucidate the complex phenomenon of RNAa/RNAi in the RNAome. This warrants a proper understanding of the RNAi/RNAa molecular pathways in living organisms before any of the small dsRNAs can potentially be exploited for therapeutics in human beings.     

The Use of NanoTrap Particles as a Sample Enrichment Method to Enhance the Detection of Rift Valley Fever Virus

Background

Rift Valley Fever Virus (RVFV) is a zoonotic virus that is not only an emerging pathogen but is also considered a biodefense pathogen due to the threat it may cause to public health and national security. The current state of diagnosis has led to misdiagnosis early on in infection. Here we describe the use of a novel sample preparation technology, NanoTrap particles, to enhance the detection of RVFV. Previous studies demonstrated that NanoTrap particles lead to both 100 percent capture of protein analytes as well as an improvement of more than 100-fold in sensitivity compared to existing methods. Here we extend these findings by demonstrating the capture and enrichment of viruses.

Results

Screening of NanoTrap particles indicated that one particle, NT53, was the most efficient at RVFV capture as demonstrated by both qRT-PCR and plaque assays. Importantly, NT53 capture of RVFV resulted in greater than 100-fold enrichment from low viral titers when other diagnostics assays may produce false negatives. NT53 was also capable of capturing and enhancing RVFV detection from serum samples. RVFV that was inactivated through either detergent or heat treatment was still found bound to NT53, indicating the ability to use NanoTrap particles for viral capture prior to transport to a BSL-2 environment. Furthermore, both NP-40-lysed virus and purified RVFV RNA were bound by NT53. Importantly, NT53 protected viral RNA from RNase A degradation, which was not observed with other commercially available beads. Incubation of RVFV samples with NT53 also resulted in increased viral stability as demonstrated through preservation of infectivity at elevated temperatures. Finally, NanoTrap particles were capable of capturing VEEV and HIV, demonstrating the broad applicability of NanoTrap particles for viral diagnostics.

Conclusion

This study demonstrates NanoTrap particles are capable of capturing, enriching, and protecting RVFV virions. Furthermore, the use of NanoTrap particles can be extended to a variety of viruses, including VEEV and HIV.