Imaging of RNA in situ hybridization by atomic force microscopy

In this study we investigated the possibility of imaging internal cellular molecules after cytochemical detection with atomic force microscopy (AFM). To this end, rat 9G and HeLa cells were hybridized with haptenized probes for 28S ribosomal RNA, human elongation factor mRNA and cytomegalovirus immediate early antigen mRNA. The haptenized hybrids were subsequently detected with a peroxidase-labelled antibody and visualized with 3,3'-diaminobenzidine (DAB). The influence of various scanning conditions on cell morphology and visibility of the signal was investigated. In order to determine the influence of ethanol dehydration on cellular structure and visibility of the DAB precipitate, cells were kept in phosphate-buffered saline (PBS) and scanned under fluid after DAB development or dehydrated and subsequently scanned dry or submerged in PBS. Direct information on the increase in height of cellular structures because of internally precipitated DAB and the height of mock-hybridized cells was available. Results show that internal DAB precipitate can be detected by AFM, with the highest sensitivity in the case of dry cells. Although a relatively large amount of DAB had to be precipitated inside the cell before it was visible by AFM, the resolution of AFM for imaging of RNA–in situ hybridization signals was slightly better than that of conventional optical microscopy. Furthermore, it is concluded that dehydration of the cells has irreversible effects on cellular structure. Therefore, scanning under fluid of previously dehydrated samples cannot be considered as a good representation of the situation before dehydration.\ud \u

PRC1‐labeled microtubule bundles and kinetochore pairs show one‐to‐one association in metaphase

In the mitotic spindle, kinetochore microtubules form k‐fibers, whereas overlap or interpolar microtubules form antiparallel arrays containing the cross‐linker protein regulator of cytokinesis 1 (PRC1). We have recently shown that an overlap bundle, termed bridging fiber, links outermost sister k‐fibers. However, the relationship between overlap bundles and k‐fibers throughout the spindle remained unknown. Here, we show that in a metaphase spindle more than 90% of overlap bundles act as a bridge between sister k‐fibers. We found that the number of PRC1‐GFP‐labeled bundles per spindle is nearly the same as the number of kinetochore pairs. Live‐cell imaging revealed that kinetochore movement in the equatorial plane of the spindle is highly correlated with the movement of the coupled PRC1‐GFP‐labeled fiber, whereas the correlation with other fibers decreases with increasing distance. Analysis of endogenous PRC1 localization confirmed the results obtained with PRC1‐GFP. PRC1 knockdown reduced the bridging fiber thickness and interkinetochore distance throughout the spindle, suggesting a function of PRC1 in bridging microtubule organization and force balance in the metaphase spindle

Multimodal wide-field two-photon excitation imaging: characterization of the technique for in vivo applications

We report fast, non-scanning, wide-field two-photon fluorescence excitation with spectral and lifetime detection for in vivo biomedical applications. We determined the optical characteristics of the technique, developed a Gaussian flat-field correction method to reduce artifacts resulting from non-uniform excitation such that contrast is enhanced, and showed that it can be used for ex vivo and in vivo cellular-level imaging. Two applications were demonstrated: (i) ex vivo measurements of beta-amyloid plaques in retinas of transgenic mice, and (ii) in vivo imaging of sulfonated gallium(III) corroles injected into tumors. We demonstrate that wide-field two photon fluorescence excitation with flat-field correction provides more penetration depth as well as better contrast and axial resolution than the corresponding one-photon wide field excitation for the same dye. Importantly, when this technique is used together with spectral and fluorescence lifetime detection modules, it offers improved discrimination between fluorescence from molecules of interest and autofluorescence, with higher sensitivity and specificity for in vivo applications

Microarrays as a diagnostic tool in prenatal screening strategies: ethical reflection

Obstetric

Intrachromosomal homologous recombination between inverted amplicons on opposing Y-chromosome arms

Amplicons – large, nearly identical repeats in direct or inverted orientation – are abundant in the male-specific region of the human Y chromosome (MSY) and provide targets for intrachromosomal non-allelic homologous recombination (NAHR). Thus far, NAHR events resulting in deletions, duplications, inversions, or isodicentric chromosomes have been reported only for amplicon pairs located exclusively on the short arm (Yp) or the long arm (Yq). Here we report our finding of four men with Y chromosomes that evidently formed by intrachromosomal NAHR between inverted repeat pairs comprising one amplicon on Yp and one amplicon on Yq. In two men with spermatogenic failure, sister-chromatid crossing-over resulted in pseudoisoYp chromosome formation and loss of distal Yq. In two men with normal spermatogenesis, intrachromatid crossing-over generated pericentric inversions. These findings highlight the recombinogenic nature of the MSY, as intrachromosomal NAHR occurs for nearly all Y-chromosome amplicon pairs, even those located on opposing chromosome arms.National Institutes of Health (U.S.)Netherlands Organization for Scientific ResearchAcademic Medical Center (University of Amsterdam

Fetal fraction of cell-free DNA in noninvasive prenatal testing and adverse pregnancy outcomes:a nationwide retrospective cohort study of 56,110 pregnant women

Background: Noninvasive prenatal testing by cell-free DNA analysis is offered to pregnant women worldwide to screen for fetal aneuploidies. In noninvasive prenatal testing, the fetal fraction of cell-free DNA in the maternal circulation is measured as a quality control parameter. Given that fetal cell-free DNA originates from the placenta, the fetal fraction might also reflect placental health and maternal pregnancy adaptation. Objective: This study aimed to assess the association between the fetal fraction and adverse pregnancy outcomes. Study Design: We performed a retrospective cohort study of women with singleton pregnancies opting for noninvasive prenatal testing between June 2018 and June 2019 within the Dutch nationwide implementation study (Trial by Dutch Laboratories for Evaluation of Non-Invasive Prenatal Testing [TRIDENT]-2). Multivariable logistic regression analysis was used to assess associations between fetal fraction and adverse pregnancy outcomes. Fetal fraction was assessed as a continuous variable and as &lt;10th percentile, corresponding to a fetal fraction &lt;2.5%. Results: The cohort comprised 56,110 pregnancies. In the analysis of fetal fraction as a continuous variable, a decrease in fetal fraction was associated with increased risk of hypertensive disorders of pregnancy (adjusted odds ratio, 2.27 [95% confidence interval, 1.89–2.78]), small for gestational age neonates &lt;10th percentile (adjusted odds ratio, 1.37 [1.28–1.45]) and &lt;2.3rd percentile (adjusted odds ratio, 2.63 [1.96–3.57]), and spontaneous preterm birth from 24 to 37 weeks of gestation (adjusted odds ratio, 1.02 [1.01–1.03]). No association was found for fetal congenital anomalies (adjusted odds ratio, 1.02 [1.00–1.04]), stillbirth (adjusted odds ratio, 1.02 [0.96–1.08]), or neonatal death (adjusted odds ratio, 1.02 [0.96–1.08]). Similar associations were found for adverse pregnancy outcomes when fetal fraction was &lt;10th percentile. Conclusion: In early pregnancy, a low fetal fraction is associated with increased risk of adverse pregnancy outcomes. These findings can be used to expand the potential of noninvasive prenatal testing in the future, enabling the prediction of pregnancy complications and facilitating tailored pregnancy management through intensified monitoring or preventive measures.</p

Fetal fraction of cell-free DNA in noninvasive prenatal testing and adverse pregnancy outcomes:a nationwide retrospective cohort study of 56,110 pregnant women

Background: Noninvasive prenatal testing by cell-free DNA analysis is offered to pregnant women worldwide to screen for fetal aneuploidies. In noninvasive prenatal testing, the fetal fraction of cell-free DNA in the maternal circulation is measured as a quality control parameter. Given that fetal cell-free DNA originates from the placenta, the fetal fraction might also reflect placental health and maternal pregnancy adaptation. Objective: This study aimed to assess the association between the fetal fraction and adverse pregnancy outcomes. Study Design: We performed a retrospective cohort study of women with singleton pregnancies opting for noninvasive prenatal testing between June 2018 and June 2019 within the Dutch nationwide implementation study (Trial by Dutch Laboratories for Evaluation of Non-Invasive Prenatal Testing [TRIDENT]-2). Multivariable logistic regression analysis was used to assess associations between fetal fraction and adverse pregnancy outcomes. Fetal fraction was assessed as a continuous variable and as &lt;10th percentile, corresponding to a fetal fraction &lt;2.5%. Results: The cohort comprised 56,110 pregnancies. In the analysis of fetal fraction as a continuous variable, a decrease in fetal fraction was associated with increased risk of hypertensive disorders of pregnancy (adjusted odds ratio, 2.27 [95% confidence interval, 1.89–2.78]), small for gestational age neonates &lt;10th percentile (adjusted odds ratio, 1.37 [1.28–1.45]) and &lt;2.3rd percentile (adjusted odds ratio, 2.63 [1.96–3.57]), and spontaneous preterm birth from 24 to 37 weeks of gestation (adjusted odds ratio, 1.02 [1.01–1.03]). No association was found for fetal congenital anomalies (adjusted odds ratio, 1.02 [1.00–1.04]), stillbirth (adjusted odds ratio, 1.02 [0.96–1.08]), or neonatal death (adjusted odds ratio, 1.02 [0.96–1.08]). Similar associations were found for adverse pregnancy outcomes when fetal fraction was &lt;10th percentile. Conclusion: In early pregnancy, a low fetal fraction is associated with increased risk of adverse pregnancy outcomes. These findings can be used to expand the potential of noninvasive prenatal testing in the future, enabling the prediction of pregnancy complications and facilitating tailored pregnancy management through intensified monitoring or preventive measures.</p