Extracellular Matrix Modulates Outgrowth Dynamics in Ovarian Cancer.

Ovarian carcinoma (OC) forms outgrowths that extend from the outer surface of an afflicted organ into the peritoneum. OC outgrowth formation is poorly understood due to the limited availability of cell culture models examining the behavior of cells that form outgrowths. Prompted by immunochemical evaluation of extracellular matrix (ECM) components in human tissues, laminin and collagen-rich ECM-reconstituted cell culture models amenable to studies of cell clusters that can form outgrowths are developed. It is demonstrated that ECM promotes outgrowth formation in fallopian tube non-ciliated epithelial cells (FNE) expressing mutant p53 and various OC cell lines. Outgrowths are initiated by cells that underwent outward translocation and retained the ability to intercalate into mesothelial cell monolayers. Electron microscopy, optical coherence tomography, and small amplitude oscillatory shear experiments reveal that increased ECM levels led to increased fibrous network thickness and high shear elasticity of the microenvironment. These physical characteristics are associated with outgrowth suppression. The low ECM microenvironment mimicks the viscoelasticity of malignant peritoneal fluid (ascites) and supports cell proliferation, cell translocation, and outgrowth formation. These results highlight the importance of the ECM microenvironment in modulating OC growth and can provide additional insights into the mode of dissemination of primary and recurrent ovarian tumors.

Serum visfatin as a metabolic biomarker in obese patients with gestational diabetes mellitus.

BACKGROUND: Visfatin is an adipokine produced and secreted by the adipose tissue. It exerts an insulin-like effect by the insulin receptor-1 and has a hypoglycemic effect. We aimed to investigate how serum visfatin changes in women with gestational diabetes mellitus (GDM), and whether it is predictive of neonatal outcomes. METHODS: Visfatin levels were prospectively measured in peripheral blood serum by enzyme immunoassay in 210 pregnant women, 156 of which were diagnosed with GDM, 18 of which suffered from pregnancy-induced hypertension (PIH) and 36 healthy controls. RESULTS: Patients with obesity class II (median=2.562 ng/mL) and class III (median=6.2940 ng/mL) had higher serum visfatin than overweight patients (median=0.735 ng/mL); (Mann-Whitney U test, P=0.037 and P=0.023, respectively). In GDM patients with BMI above 30, serum visfatin was associated to glycosylated hemoglobin (Spearman correlation test, R=0.26, P=0.045). Women with BMI above 25 treated with insulin had lower serum visfatin levels than those treated with diet only (Mann-Whitney U test, P=0.045). No correlation was found between visfatin and parameters of lipid profile such as HDL, LDL, or triglycerides (Spearman correlation tests, R=-0.051, -0.1, 0.0019; P=0.54, 0.29, 0.98, respectively). We observed that visfatin was not associated with birth weight (Spearman correlation test, R=-0.014, P=0.86) or adverse neonatal outcome as measured by umbilical artery pH below 7.25 (Mann-Whitney U test, P=0.55) or Apgar score below 10 (Mann-Whitney U test, P=0.21). CONCLUSIONS: In GDM patients with higher BMI, serum visfatin was elevated, correlated positively with glycosylated hemoglobin, and decreased upon treatment with insulin therapy.

High levels of AAV vector integration into CRISPR-induced DNA breaks.

Adeno-associated virus (AAV) vectors have shown promising results in preclinical models, but the genomic consequences of transduction with AAV vectors encoding CRISPR-Cas nucleases is still being examined. In this study, we observe high levels of AAV integration (up to 47%) into Cas9-induced double-strand breaks (DSBs) in therapeutically relevant genes in cultured murine neurons, mouse brain, muscle and cochlea. Genome-wide AAV mapping in mouse brain shows no overall increase of AAV integration except at the CRISPR/Cas9 target site. To allow detailed characterization of integration events we engineer a miniature AAV encoding a 465 bp lambda bacteriophage DNA (AAV-λ465), enabling sequencing of the entire integrated vector genome. The integration profile of AAV-465λ in cultured cells display both full-length and fragmented AAV genomes at Cas9 on-target sites. Our data indicate that AAV integration should be recognized as a common outcome for applications that utilize AAV for genome editing.

Allele-specific gene editing prevents deafness in a model of dominant progressive hearing loss.

Since most dominant human mutations are single nucleotide substitutions1,2, we explored gene editing strategies to disrupt dominant mutations efficiently and selectively without affecting wild-type alleles. However, single nucleotide discrimination can be difficult to achieve3 because commonly used endonucleases, such as Streptococcus pyogenes Cas9 (SpCas9), can tolerate up to seven mismatches between guide RNA (gRNA) and target DNA. Furthermore, the protospacer-adjacent motif (PAM) in some Cas9 enzymes can tolerate mismatches with the target DNA3,4. To circumvent these limitations, we screened 14 Cas9/gRNA combinations for specific and efficient disruption of a nucleotide substitution that causes the dominant progressive hearing loss, DFNA36. As a model for DFNA36, we used Beethoven mice5, which harbor a point mutation in Tmc1, a gene required for hearing that encodes a pore-forming subunit of mechanosensory transduction channels in inner-ear hair cells6. We identified a PAM variant of Staphylococcus aureus Cas9 (SaCas9-KKH) that selectively and efficiently disrupted the mutant allele, but not the wild-type Tmc1/TMC1 allele, in Beethoven mice and in a DFNA36 human cell line. Adeno-associated virus (AAV)-mediated SaCas9-KKH delivery prevented deafness in Beethoven mice up to one year post injection. Analysis of current ClinVar entries revealed that ~21% of dominant human mutations could be targeted using a similar approach.

CRISPR/Cas9 Mediated Disruption of the Swedish APP Allele as a Therapeutic Approach for Early-Onset Alzheimer's Disease.

The APPswe (Swedish) mutation in the amyloid precursor protein (APP) gene causes dominantly inherited Alzheimer's disease (AD) as a result of increased ß-secretase cleavage of the amyloid-ß (Aß) precursor protein. This leads to abnormally high Aß levels, not only in brain but also in peripheral tissues of mutation carriers. Here, we selectively disrupted the human mutant APPSW allele using CRISPR. By applying CRISPR/Cas9 from Streptococcus pyogenes, we generated allele-specific deletions of either APPSW or APPWT. As measured by ELISA, conditioned media of targeted patient-derived fibroblasts displayed an approximate 60% reduction in secreted Aß. Next, coding sequences for the APPSW-specific guide RNA (gRNA) and Cas9 were packaged into separate adeno-associated viral (AAV) vectors. Site-specific indel formation was achieved both in primary neurons isolated from APPSW transgenic mouse embryos (Tg2576) and after co-injection of these vectors into hippocampus of adult mice. Taken together, we here present proof-of-concept data that CRISPR/Cas9 can selectively disrupt the APPSW allele both ex vivo and in vivo-and thereby decrease pathogenic Aß. Hence, this system may have the potential to be developed as a tool for gene therapy against AD caused by APPswe and other point mutations associated with increased Aß.

Extracellular Vesicles: Composition, Biological Relevance, and Methods of Study.

The release of extracellular vesicles (EVs), including exosomes and microvesicles, is a phenomenon shared by many cell types as a means of communicating with other cells and also potentially removing cell contents. The cargo of EVs includes the proteins, lipids, nucleic acids, and membrane receptors of the cells from which they originate. EVs released into the extracellular space can enter body fluids and potentially reach distant tissues. Once taken up by neighboring and/or distal cells, EVs can transfer functional cargo that may alter the status of recipient cells, thereby contributing to both physiological and pathological processes. In this article, we will focus on EV composition, mechanisms of uptake, and their biological effects on recipient cells. We will also discuss established and recently developed methods used to study EVs, including isolation, quantification, labeling and imaging protocols, as well as RNA analysis.