Fecundity and early life of the deep-water jellyfish Periphylla periphylla

Comparisons over 6 years of three Norwegian fjord populations of the deep-water scyphomedusa Periphylla periphylla are presented. A minor part of the population in Lurefjord is migrating to the surface during night, which benefits mating encounters by increasing abundance per unit volume and decreasing the distance between individuals. Simulations using a typical water-column density profile and Stoke’s law show that fertilized eggs released in the surface quickly reach a depth where light is insufficient for visual predators. Consequently, the distribution of the smallest juveniles was strongly skewed towards higher depths in all three fjords studied. Mature females in Sognefjord were 4–5 times less abundant than in Lurefjord and Halsafjord, but due to a larger size and strong exponential relationship between size and number of mature oocytes, the potential recruitment rate as recruits m−2 year−1 was not much different from the other two fjords. Nevertheless, the observed number of small (<1 cm) juveniles was 18–31 times higher in Sognefjord than in the other two fjords, and it is assumed that the deeper habitat (up to 1300 m) compared to the other fjords (up to 440 and 530 m) is a superior habitat for the early development of P. periphylla.publishedVersio

Dissecting the role of distinct OCT4-SOX2 heterodimer configurations in pluripotency

The transcription factors OCT4 and SOX2 are required for generating induced pluripotent stem cells (iPSCs) and for maintaining embryonic stem cells (ESCs). OCT4 and SOX2 associate and bind to DNA in different configurations depending on the arrangement of their individual DNA binding elements. Here we have investigated the role of the different OCT4-SOX2-DNA assemblies in regulating and inducing pluripotency. To this end, we have generated SOX2 mutants that interfere with specific OCT4-SOX2 heterodimer configurations and assessed their ability to generate iPSCs and to rescue ESC self-renewal. Our results demonstrate that the OCT4-SOX2 configuration that dimerizes on a Hoxb1-like composite, a canonical element with juxtaposed individual binding sites, plays a more critical role in the induction and maintenance of pluripotency than any other OCT4-SOX2 configuration. Overall, the results of this study provide new insight into the protein interactions required to establish a de novo pluripotent network and to maintain a true pluripotent cell fate.Link_to_subscribed_fulltex

An insulin hypersecretion phenotype precedes pancreatic β cell failure in MODY3 patient-specific cells

MODY3 is a monogenic hereditary form of diabetes caused by mutations in the transcription factor HNF1A. The patients progressively develop hyperglycemia due to perturbed insulin secretion, but the pathogenesis is unknown. Using patient-specific hiPSCs, we recapitulate the insulin secretion sensitivity to the membrane depolarizing agent sulfonylurea commonly observed in MODY3 patients. Unexpectedly, MODY3 patient-specific HNF1A+/R272C β cells hypersecrete insulin both in vitro and in vivo after transplantation into mice. Consistently, we identified a trend of increased birth weight in human HNF1A mutation carriers compared with healthy siblings. Reduced expression of potassium channels, specifically the KATP channel, in MODY3 β cells, increased calcium signaling, and rescue of the insulin hypersecretion phenotype by pharmacological targeting ATP-sensitive potassium channels or low-voltage-activated calcium channels suggest that more efficient membrane depolarization underlies the hypersecretion of insulin in MODY3 β cells. Our findings identify a pathogenic mechanism leading to β cell failure in MODY3.Peer reviewe

Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo

While small interfering RNAs (siRNAs) have been rapidly appreciated to silence genes, efficient and non-toxic vectors for primary cells and for systemic in vivo delivery are lacking. Several siRNA-delivery vehicles, including cell-penetrating peptides (CPPs), have been developed but their utility is often restricted by entrapment following endocytosis. Hence, developing CPPs that promote endosomal escape is a prerequisite for successful siRNA implementation. We here present a novel CPP, PepFect 6 (PF6), comprising the previously reported stearyl-TP10 peptide, having pH titratable trifluoromethylquinoline moieties covalently incorporated to facilitate endosomal release. Stable PF6/siRNA nanoparticles enter entire cell populations and rapidly promote endosomal escape, resulting in robust RNAi responses in various cell types (including primary cells), with minimal associated transcriptomic or proteomic changes. Furthermore, PF6-mediated delivery is independent of cell confluence and, in most cases, not significantly hampered by serum proteins. Finally, these nanoparticles promote strong RNAi responses in different organs following systemic delivery in mice without any associated toxicity. Strikingly, similar knockdown in liver is achieved by PF6/siRNA nanoparticles and siRNA injected by hydrodynamic infusion, a golden standard technique for liver transfection. These results imply that the peptide, in addition to having utility for RNAi screens in vitro, displays therapeutic potential

Fecundity and early life of the deep-water jellyfish Periphylla periphylla

Comparisons over 6 years of three Norwegian fjord populations of the deep-water scyphomedusa Periphylla periphylla are presented. A minor part of the population in Lurefjord is migrating to the surface during night, which benefits mating encounters by increasing abundance per unit volume and decreasing the distance between individuals. Simulations using a typical water-column density profile and Stoke’s law show that fertilized eggs released in the surface quickly reach a depth where light is insufficient for visual predators. Consequently, the distribution of the smallest juveniles was strongly skewed towards higher depths in all three fjords studied. Mature females in Sognefjord were 4–5 times less abundant than in Lurefjord and Halsafjord, but due to a larger size and strong exponential relationship between size and number of mature oocytes, the potential recruitment rate as recruits m−2 year−1 was not much different from the other two fjords. Nevertheless, the observed number of small (<1 cm) juveniles was 18–31 times higher in Sognefjord than in the other two fjords, and it is assumed that the deeper habitat (up to 1300 m) compared to the other fjords (up to 440 and 530 m) is a superior habitat for the early development of P. periphylla

Epigenetic Aberrations Are Not Specific to Transcription Factor-Mediated Reprogramming

Somatic cells can be reprogrammed to pluripotency using different methods. In comparison with pluripotent cells obtained through somatic nuclear transfer, induced pluripotent stem cells (iPSCs) exhibit a higher number of epigenetic errors. Furthermore, most of these abnormalities have been described to be intrinsic to the iPSC technology. Here, we investigate whether the aberrant epigenetic patterns detected in iPSCs are specific to transcription factor-mediated reprogramming. We used germline stem cells (GSCs), which are the only adult cell type that can be converted into pluripotent cells (gPSCs) under defined culture conditions, and compared GSC-derived iPSCs and gPSCs at the transcriptional and epigenetic level. Our results show that both reprogramming methods generate indistinguishable states of pluripotency. GSC-derived iPSCs and gPSCs retained similar levels of donor cell-type memory and exhibited comparable numbers of reprogramming errors. Therefore, our study demonstrates that the epigenetic abnormalities detected in iPSCs are not specific to transcription factor-mediated reprogramming

In-Silico Staining from Bright-Field and Fluorescent Images Using Deep Learning

Fluorescent markers are commonly used to characterize single cells and to uncover molecular properties. Unfortunately, fluorescent staining is laborious and costly, it damages tissue and suffers from inconsistencies. Recently deep learning approaches have been successfully applied to predict fluorescent markers from bright-field images [1&ndash;3]. These approaches can save costs and time and speed up the classification of tissue properties. However, it is currently not clear how different image channels can be meaningfully combined to improve prediction accuracy. Thus, we investigated the benefits of multi channel input for predicting a specific transcription factor antibody staining. Our image dataset consists of three channels: bright-field, fluorescent GFP reporter and transcription factor antibody staining. Fluorescent GFP is constantly expressed in the genetically modified cells from a particular differentiation step onwards. The cells are additionally stained with a specific transcription factor antibody that marks a subtype of GFP positive cells. For data acquisition we used a Leica SP8 and a Zeiss LSM780 microscope with 20x objectives. We trained a deep neural network, a modified U-Net [4], to predict the transcription factor antibody staining from bright-field and GFP channels. To this end, we trained on 2432 three-dimensional images containing roughly 7600 single cells and compared the accuracy for prediction of the transcription factor antibody staining using bright-field only, GFP only, and both channels together on a test-set of 576 images with approximately 1800 single cells. The same training- and test-set was used for all experiments (Fig.&nbsp;1). The prediction error, measured as the mean relative pixel-wise error over the test-set, was calculated to 61% for prediction from bright-field, 55% for prediction from GFP and 51% for prediction both bright-field and GFP images. The median pixel-wise Pearson correlation coefficient, increases from 0.12 for prediction from bright-field channels to 0.17 for prediction from GFP channels, to 0.31 for prediction from bright-field and GFP channels (Fig.&nbsp;2). Our work demonstrates that prediction performance can be increased by combining multiple channels for in-silico prediction of stainings. We anticipate this research to be a starting point for further investigations on which stainings could be predicted from other stainings using deep learning. These approaches bear a huge potential in saving laborious and costly work for researchers and clinical technicians and could reveal biological relationships between fluorescent markers

Counteracting Activities of OCT4 and KLF4 during Reprogramming to Pluripotency

Differentiated cells can be reprogrammed into induced pluripotent stem cells (iPSCs) after overexpressing four transcription factors, of which Oct4 is essential. To elucidate the role of Oct4 during reprogramming, we investigated the immediate transcriptional response to inducible Oct4 overexpression in various somatic murine cell types using microarray analysis. By downregulating somatic-specific genes, Oct4 induction influenced each transcriptional program in a unique manner. A significant upregulation of pluripotent markers could not be detected. Therefore, OCT4 facilitates reprogramming by interfering with the somatic transcriptional network rather than by directly initiating a pluripotent gene-expression program. Finally, Oct4 overexpression upregulated the gene Mgarp in all the analyzed cell types. Strikingly, Mgarp expression decreases during the first steps of reprogramming due to a KLF4-dependent inhibition. At later stages, OCT4 counteracts the repressive activity of KLF4, thereby enhancing Mgarp expression. We show that this temporal expression pattern is crucial for the efficient generation of iPSCs

Reprogramming to Pluripotency through a Somatic Stem Cell Intermediate

<div><p>Transcription factor-based reprogramming can lead to the successful switching of cell fates. We have recently reported that mouse embryonic fibroblasts (MEFs) can be directly reprogrammed into induced neural stem cells (iNSCs) after the forced expression of <i>Brn4</i>, <i>Sox2</i>, <i>Klf4</i>, and <i>Myc</i>. Here, we tested whether iNSCs could be further reprogrammed into induced pluripotent stem cells (iPSCs). The two factors <i>Oct4</i> and <i>Klf4</i> were sufficient to induce pluripotency in iNSCs. Immunocytochemistry and gene expression analysis showed that iNSC-derived iPSCs (iNdiPSCs) are similar to embryonic stem cells at the molecular level. In addition, iNdiPSCs could differentiate into cells of all three germ layers, both <i>in vitro</i> and <i>in vivo</i>, proving that iNdiPSCs are <i>bona fide</i> pluripotent cells. Furthermore, analysis of the global gene expression profile showed that iNdiPSCs, in contrast to iNSCs, do not retain any MEF transcriptional memory even at early passages after reprogramming. Overall, our results demonstrate that iNSCs can be reprogrammed to pluripotency and suggest that cell fate can be redirected numerous times. Importantly, our findings indicate that the induced pluripotent cell state may erase the donor-cell type epigenetic memory more efficiently than other induced somatic cell fates.</p> </div

iNdiPSCs can differentiate into cells of all three germ layers <i>in</i><i>vitro</i> and <i>in</i><i>vivo</i>.

<p>(<b>A</b>) Immunofluorescence microscopy images of iNdiPSC-1 and -2-differentiated embryoid bodies using antibodies directed against TUBB3/β3-TUBULIN (ectoderm), SOX17 (endoderm), and ACTA2 (α-SMA) (mesoderm). Scale bars, 150 µm. (<b>B</b>) Teratoma analysis of iNdiPSC-1 and -2 showing tissues of all three germ layers: ectoderm (neural rosette and keratinocytes), endoderm (gut-like endothelium), and mesoderm (muscle and cartilage).</p