The central vision-threatening event in glaucoma is dysfunction and loss of retinal ganglion cells (RGCs), thought to be promoted by local tissue deformations. Here, we sought to reduce tissue ...deformation near the optic nerve head by selectively stiffening the peripapillary sclera, i.e. the scleral region immediately adjacent to the optic nerve head. Previous scleral stiffening studies to treat glaucoma or myopia have used either pan-scleral stiffening (not regionally selective) or regionally selective stiffening with limited access to the posterior globe. We present a method for selectively stiffening the peripapillary sclera using a transpupillary annular light beam to activate methylene blue administered by retrobulbar injection. Unlike prior approaches to photocrosslinking in the eye, this approach avoids the damaging effects of ultraviolet light by employing red light. This targeted photocrosslinking approach successfully stiffened the peripapillary sclera at 6 weeks post-treatment, as measured by whole globe inflation testing. Specifically, strain was reduced by 47% when comparing treated vs. untreated sclera within the same eye (n = 7, p=0.0064) and by 54% when comparing the peripapillary sclera of treated vs. untreated eyes (n = 7, p<0.0001). Post-treatment characterization of RGCs (optic nerve axon counts/density, and grading), retinal function (electroretinography), and retinal histology revealed that photocrosslinking was associated with some ocular toxicity. We conclude that a transpupillary photocrosslinking approach enables selective scleral stiffening targeted to the peripapillary region that may be useful in future treatments of glaucoma.
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•Localized stiffening of the posterior eye may be a treatment strategy for glaucoma and myopia.•We sought to photocrosslink scleral collagen, using methylene blue as a photoinitiator, in vivo.•We stiffened an annular region around the optic nerve with a transpupillary red light beam.•Moderate treatment toxicity was identified using axon counts, histology, and electroretinography.
Skeletal muscle differentiation entails the coordination of muscle-specific gene expression and terminal withdrawal from the cell cycle. This cell cycle arrest in the G$_0$ phase requires the ...retinoblastoma tumor suppressor protein (Rb). The function of Rb is negatively regulated by cyclin-dependent kinases (Cdks), which are controlled by Cdk inhibitors. Expression of MyoD, a skeletal muscle-specific transcriptional regulator, activated the expression of the Cdk inhibitor p21 during differentiation of murine myocytes and in nonmyogenic cells. MyoD-mediated induction of p21 did not require the tumor suppressor protein p53 and correlated with cell cycle withdrawal. Thus, MyoD may induce terminal cell cycle arrest during skeletal muscle differentiation by increasing the expression of p21.
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The implementation of the proposal from the European Chemical Agency (ECHA) to restrict the use of nanoplastics (NP) and microplastics (MP) in consumer products will require reliable ...methods to perform size and mass-based concentration measurements. Analytical challenges arise at the nanometre to micrometre interface, e.g., 800 nm–10 µm, where techniques applicable at the nanometre scale reach their upper limit of applicability and approaches applicable at the micrometre scale must be pushed to their lower limits of detection.
Herein, we compared the performances of nine analytical techniques by measuring the particle size distribution and mass-based concentration of polystyrene mixtures containing both nano and microparticles, with the educational aim to underline applicability and limitations of each technique.
Light scattering-based measurements do not have the resolution to distinguish multiple populations in polydisperse samples. Nanoparticle tracking analysis (NTA), nano-flowcytometry (nFCM) and asymmetric flow field flow fractionation hyphenated with multiangle light scattering (AF4-MALS) cannot measure particles in the micrometre range. Static light scattering (SLS) is not able to accurately detect particles below 200 nm, and similarly to transmission electron microscopy (TEM) and flow cytometry (FCM), is not suitable for accurate mass-based concentration measurements. Alternatives for high-resolution sizing and concentration measurements in the size range between 60 nm and 5 µm are tunable resistive pulse sensing (TRPS) and centrifugal liquid sedimentation (CLS), that can bridge the gap between the nanometre and micrometre range.
Complex regulatory networks regulate stem cell behavior and contributions to tissue growth, repair, and homeostasis. A full understanding of the networks controlling stem cell self-renewal and ...differentiation, however, has not yet been realized. To systematically dissect these networks and identify their components, we performed an unbiased, transcriptome-wide in vivo RNAi screen in female Drosophila germline stem cells (GSCs). Based on characterized cellular defects, we classified 646 identified genes into phenotypic and functional groups and unveiled a comprehensive set of networks regulating GSC maintenance, survival, and differentiation. This analysis revealed an unexpected role for ribosomal assembly factors in controlling stem cell cytokinesis. Moreover, our data show that the transition from self-renewal to differentiation relies on enhanced ribosome biogenesis accompanied by increased protein synthesis. Collectively, these results detail the extensive genetic networks that control stem cell homeostasis and highlight the intricate regulation of protein synthesis during differentiation.
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•An in vivo RNAi screen identifies 646 genes involved in germline stem cell biology•Ribosome assembly and eIF4E modulate the final step of stem cell cytokinesis•Stem cell differentiation relies on pathways promoting global protein synthesis•rRNA and protein synthesis are uncoupled during stem cell differentiation
The complex gene regulatory networks that control stem cell behavior are only partially understood. Sanchez et al. describe an unbiased in vivo RNAi screen and provide a formative dissection of the gene pathways required for germline stem cell development.
Mature microRNAs (miRNAs) are generated via a two-step processing pathway to yield approximately 22-nucleotide small RNAs that regulate gene expression at the post-transcriptional level. Initial ...cleavage is catalysed by Drosha, a nuclease of the RNase III family, which acts on primary miRNA transcripts (pri-miRNAs) in the nucleus. Here we show that Drosha exists in a multiprotein complex, the Microprocessor, and begin the process of deconstructing that complex into its constituent components. Along with Drosha, the Microprocessor also contains Pasha (partner of Drosha), a double-stranded RNA binding protein. Suppression of Pasha expression in Drosophila cells or Caenorhabditis elegans interferes with pri-miRNA processing, leading to an accumulation of pri-miRNAs and a reduction in mature miRNAs. Finally, depletion or mutation of pash-1 in C. elegans causes de-repression of a let-7 reporter and the appearance of phenotypic defects overlapping those observed upon examination of worms with lesions in Dicer (dcr-1) or Drosha (drsh-1). Considered together, these results indicate a role for Pasha in miRNA maturation and miRNA-mediated gene regulation.
This analysis examines the association between maternal characteristics, particularly body mass index (BMI) and infant birth weight in 1048 live infants. Mean reported pre pregnancy BMI of mothers ...was 23.74 kg/m2 (SD 4.21). The educational level of the mother's parents was independently associated with maternal BMI, those with higher educated parents having a lower reported BMI (F = 2.787, p = 0.029). Mean infant birth weight was 3493 g (SD 18.1) and there was a strong graduated relationship to estimated gestational age. In a sub-group of participating maternal grandmothers (n = 171), reported BMI was 26.7Kg/m2. The BMI of expectant mothers was significantly associated with their own mother's BMI. (r = 0.179, p = 0.005) in this sub-group. These preliminary findings, which will be investigated further with recorded height and weight information, suggest that familial factors are influential, perhaps through genetic predisposition or shared socio-cultural factors such as diet.
We have used high real-space resolution pulsed neutron diffraction to investigate the structure of fibre-forming Ge-based multicomponent sulphide glasses. Diffraction patterns were measured up to a ...maximum momentum transfer of 40 Å
−1 for a series of seven samples from the stoichiometric system Ge
25(As,Ga)
10S
65, with Ga contents from 0 to 10 at.%. The correlation functions all have three principal peaks at 2.23, 2.97 and 3.49 Å. The first principal peak in the correlation functions is predominantly due to Ge–S bonds, together with smaller contributions from As–S and Ga–S bonds. The second principal peak is due to cation–cation distances between edge-sharing structural units. The third principal peak is due mostly to S–S distances within structural units. There is also fine structure around the first peak of the correlation functions, consisting of a small `pre-peak' at ∼2.0 Å and a larger and broader shoulder at ∼2.5 Å. There are no detectable changes with composition in the pre-peak, whilst the shoulder grows as gallium is added to the glass. The position of the pre-peak, 2.0 Å, is consistent with either S
2
2− disulphide groups within a structural unit, or with S–S bonds in the network, due to chemical disorder. According to the chemical disorder model, the peak at 2.5 Å is due to Ge–Ge bonds in the network. If gallium is tetrahedrally coordinated then the number of these bonds must increase as gallium replaces arsenic. Such an increase is observed in the experimental data and hence the chemical disorder model is preferred over the disulphide model.
Piwi proteins specify an animal-specific subclass of the Argonaute family that, in vertebrates, is specifically expressed in germ cells. We demonstrate that zebrafish Piwi (Ziwi) is expressed in both ...the male and the female gonad and is a component of a germline-specifying structure called nuage. Loss of Ziwi function results in a progressive loss of germ cells due to apoptosis during larval development. In animals that have reduced Ziwi function, germ cells are maintained but display abnormal levels of apoptosis in adults. In mammals, Piwi proteins associate with approximately 29-nucleotide-long, testis-specific RNA molecules called piRNAs. Here we show that zebrafish piRNAs are present in both ovary and testis. Many of these are derived from transposons, implicating a role for piRNAs in the silencing of repetitive elements in vertebrates. Furthermore, we show that piRNAs are Dicer independent and that their 3′ end likely carries a 2′O-Methyl modification.
RNA interference (RNAi) is the mechanism through which double-stranded
RNAs silence cognate genes. In plants, this can
occur at both the transcriptional and the post-transcriptional levels; however, ...in animals, only post-transcriptional RNAi has been
reported to date. In both plants and animals, RNAi is characterized by the
presence of RNAs of about 22 nucleotides in length that are homologous to
the gene that is being suppressed. These 22-nucleotide
sequences serve as guide sequences that instruct a multicomponent nuclease,
RISC, to destroy specific messenger RNAs. Here we identify
an enzyme, Dicer, which can produce putative guide RNAs. Dicer is a member
of the RNase III family of nucleases that specifically cleave double-stranded
RNAs, and is evolutionarily conserved in worms, flies, plants, fungi and mammals.
The enzyme has a distinctive structure, which includes a helicase domain and
dual RNase III motifs. Dicer also contains a region of homology to the RDE1/QDE2/ARGONAUTE
family that has been genetically linked to RNAi.
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