List of polyadenylation factors predicted to interact with RBPL-1 according to the interactome on WormBase.

<p>List of polyadenylation factors predicted to interact with RBPL-1 according to the interactome on WormBase.</p

RBPL-1 is expressed in diverse tissues and localized to the nucleolus. a.

<p>RBPL-1::GFP is expressed in multiple tissues, including neurons, intestine, spermatheca, and vulva. Scale bar: 20 µm. <b>b.</b> RBPL-1::GFP is localized to the nucleolus of intestine cells. The bottom left image indicates the nucleolar localization of RBPL-1 within an intestine nucleus (the dashed line indicates the nucleus). Scale bar: 50 µm. <b>c.</b> Dynamics of RBPL-1 during embryogenesis. A transient disappearance of RBPL-1 fluorescence occurs from metaphase to telophase. Membrane and RBPL-1 are marked by GFP, and nuclei are shown by H2B::mCherry. Scale bar: 2 µm.</p

The <em>C. elegans</em> Homolog of RBBP6 (RBPL-1) Regulates Fertility through Controlling Cell Proliferation in the Germline and Nutrient Synthesis in the Intestine

<div><p>RBBP6 (retinoblastoma binding protein 6, also known as PACT or P2P-R in humans) is a multi-domain protein that functions in multiple processes, such as mitosis, cell differentiation, and cell apoptosis. RBBP6 is evolutionarily conserved and is present in unicellular organisms to mammals. Studies of RBBP6 have mostly focused on its RB- and p53-binding domains, which are found exclusively in mammals. Here, we investigated the <i>C. elegans</i> homolog of RBBP6 to explore the functional roles of its other domains. We found that RBPL-1, the homolog of RBBP6 in <i>C. elegans</i>, is indispensable for worm development. RNAi silencing of <i>rbpl-1</i> led to embryonic lethality, as well as defects in oocyte production and intestine development. <i>rbpl-1</i> RNAi worms showed defects in germ cell proliferation, suggesting that RBPL-1 regulates mitosis. Moreover, RNAi silencing of <i>rbpl-1</i> inhibited nutrient synthesis in the worm intestine. RBPL-1, as a nucleolus protein, was found to be expressed in diverse tissues and necessary for both germline and soma development. Using microarray analysis, we identified ≈700 genes whose expression levels were changed at least 10-fold in <i>rbpl-1</i> worms. We propose that RBPL-1, like its yeast homolog, may regulate gene expression as an mRNA cleavage and polyadenylation factor. Taken together, the findings from this study reveal that RBPL-1 plays a pivotal role in <i>C. elegans</i> germline and soma development, suggesting that the functions of RBBP6 are conserved in diverse eukaryotic species.</p> </div

RNAi for <i>rbpl-1</i> reduces oocyte number. a.

<p>RNAi knockdown of <i>rbpl-1</i> results in reduced embryo number. The earlier RNAi is applied, the more severe the defects. The empty vector L4440 was used as an RNAi control. The grow temperature was 15°C. (YA = Young Adult, A = Adult). <b>b.. </b><i>rbpl-1</i> RNAi silenced hermaphrodites crossed with <i>him-5</i> males produce no more progeny than control RNAi hermaphrodites only. <b>c.</b> Adult <i>rbpl-1</i> RNAi worms show abnormal germline development. There is no difference between the <i>rbpl-1</i> RNAi and control RNAi in germline development at L3 stage. In adults, the oocytes are abnormal and reduced in <i>rbpl-1</i> worms. Some oocytes in <i>rbpl-1</i> RNAi adult possess multiple sets of chromosomes (shown in the inset) (H2B::GFP fluorescence indicates the nucleus of germ cells). Scale bar: 30 µm. <b>d.. </b><i>rbpl-1</i> RNAi in either soma or germline significantly reduces egg production. In the strain NL2098, RNAi is effective in the germline and intestine, while in the strain CB4856, RNAi is effective only in the soma. *<i>P</i><0.05.</p

RBPL-1 is conserved in <i>C. elegans</i>. a.

<p>Predicted structure of RBPL-1 (TAG-214). The conserved domains DWNN, zinc knuckle, and RING finger are indicated by blue, red, and gray boxes, respectively. The nuclear localization signal (NLS) is indicated by a green box. <b>b.</b> Protein sequence alignment highlights the conservation of the DWNN, Zn knuckle, and RING finger domains among human, rat, and <i>C. elegans</i> RBBP6 proteins. <b>c.</b> Full-length <i>rbpl-1</i> mRNA cloned by RT-PCR. <b>d.</b> RBPL-1 expressed in <i>E. coli</i>. Upper: SDS-PAGE analysis of the bacterially expressed GST-RBPL-1 fusion protein, indicated by the red arrow. Lower: western blotting analysis of GST-RBPL-1 fusion protein (the red arrow) using an anti-GST antibody.</p

RNAi for <i>rbpl-1</i> results in defects of intestine development a.

<p>The intestine (indicated by dashed line) has nearly disappeared in the <i>rbpl-1</i> worms compared with controls. Scale bar: 25 µm. <b>b.</b> Continuous RNAi of <i>rbpl-1</i> in the intestine leads to a progressively reduced brood size through generations. *<i>P</i><0.05. <b>c</b>. The production of vitellogenin (indicated by vit-2::GFP) is inhibited by RNAi silencing of <i>rbpl-1</i> (arrow head indicates the oocyte). Scale bar: 50 µm. <b>d.</b> Fluorescence imaging showing that lipid content is largely reduced upon silencing of <i>rbpl-1</i> in the whole worm (N2) or intestine only (VP303), but not in the hypodermis only (NR222). Scale bar: 25 µm. <b>e.</b> Survival time is shortened when silencing <i>rbpl-1</i> in the whole worm (N2) or only in the intestine (VP303), whereas RNAi for <i>rbpl-1</i> in the hypodermis (NR222) does not affect life span. <b>f.</b> Quantification of fluorescence intensity in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058736#pone-0058736-g004" target="_blank">Figure 4D</a> indicates that the lipid content decreases significantly when silencing <i>rbpl-1</i> in the whole worm (N2), or only in the intestine (VP303), but not when restricted to the hypodermis (NR222). *<i>P</i><0.01.</p

RNAi for <i>rbpl-1</i> causes defects of germ cell proliferation. a.

<p>The posterior end of the germline showing the mitotic and transition zones. Nuclei are labeled by H2B::GFP. Scale bar: 10 µm. <b>b.</b> Fewer mitotic cells are observed in <i>rbpl-1</i> RNAi worms than in control worms by immunofluorescence. The arrow indicates mitotic cells labeled with anti-P-H3S10. Scale bar: 10 µm. <b>c.</b> The mitotic index in <i>rbpl-1</i> RNAi worms is much lower than in control worms. Mitotic index: the number of cells in mitotic stage (staining by anti-P-H3S10) divided by the total cell number in mitotic zone (staining by DAPI).</p

Supplementary Material for: Old Problem in a New Guise: Retinal Pigment Epithelium Tear after Intravitreal Faricimab (Vabysmo^®) Injection

This case report describes a 78-year-old patient who developed a tear of the retinal pigment epithelium (RPE) during faricimab (Vabysmo®) therapy. After three consecutive intravitreal aflibercept (Eylea®) injections with persistent disease activity, therapy was switched to faricimab. The patient experienced a tear in the RPE 4 weeks postinjection. We report the first published case of RPE tear development after intravitreal faricimab injection in neovascular age-related macular degeneration. Faricimab has a new target structure in the angiopoietin-2 receptor in addition to VEGF. Patients at risk for RPE rupture were excluded from pivotal studies. Further investigation is needed to understand the effect of faricimab not only on visual acuity and intraretinal and subretinal fluid but also on mechanical stress on the RPE monolayer

Additional file 1 of Safety and efficacy comparison of polyethylene glycol, hemp seed oil, and 5% sugar brine for bowel preparation in older patients: study protocol for a randomized controlled trial

Additional file 1: SPIRIT Checklist for Trials

An efficient <i>Agrobacterium</i>-mediated soybean transformation method using green fluorescent protein as a selectable marker

Genetic transformation plays a vital role in gene functional study and molecular breeding of soybean. Conventional soybean transformation methods using chemical selectable markers, such as antibiotic or herbicide resistance genes, rely on the identification of positive transgenic lines at advanced developmental stages, making selection procedure labor intensive and time consuming. Utilization of a visual maker to track the transgene would avoid the uncertainty and blindness in the transformation process. In this research, we used green fluorescent protein (GFP) as the selectable marker to detect transgenics at early stages of soybean development. Positive transformants were detected recurrently during each stage of the process based on visualization of the green fluorescence signal, which help us to discard the non-transgenic ones in each stage to reduce the unnecessary experimental cost and lab space. In addition, the positive transgenic seeds can be identified before planting for early detection of transgene and obtain homozygous lines in advance. The method established in this study is also a useful reference for other plant species.</p