MicroRNAs in Differentiation of Embryoid Bodies and the Teratoma Subtype of Testicular Cancer

Background: Testicular germ cell tumours (TGCTs) are the most frequent tumour type among young, adult men. TGCTs can be efficiently treated, but metastases of the teratoma subtype, for which there are no circulating biomarkers, represent a challenge. Materials and Methods: Global microRNA expression in teratoma tissue and embryoid bodies was assessed using next-generation sequencing. Levels of microRNAs identified as potential biomarkers were obtained from serum of patients with teratoma and matched healthy men. Results: We identified miR-222-5p, miR-200a-5p, miR-196b-3p and miR-454-5p as biomarker candidates from the tumour tissue and embryoid body screening but the expression of these microRNAs was very low in serum and not statistically different between patients and controls. miR-375-3p was highly expressed, being highest in patients with teratoma (p=0.012) but the levels of expression in serum from these patients and healthy controls overlapped. miR-371a-3p was not expressed in serum from patients with pure teratoma, only in patients with mixed tumours. Conclusion: The microRNA profiles of the teratoma subtype of TGCT and embryoid bodies were obtained and assessed for candidate circulating biomarkers, but none with high sensitivity and specificity for teratoma were identified in our study. We conclude that neither the proposed teratoma marker miR-375-3p nor miR-371a-3p are suitable as circulating teratoma markers.publishedVersio

Reprogrammed Cells Display Distinct Proteomic SignaturesAssociated with Colony Morphology Variability

Human induced pluripotent stem cells (hiPSCs) are of high interest because they can be differentiated into a vast range of different cell types. Ideally, reprogrammed cells should sustain long-term culturing in an undifferentiated state. However, some reprogrammed cell lines represent an unstable state by spontaneously differentiating and changing their cellular phenotype and colony morphology. This phenomenon is not fully understood, and no method is available to predict it reliably. In this study, we analyzed and compared the proteome landscape of 20 reprogrammed cell lines classified as stable and unstable based on long-term colony morphology. We identified distinct proteomic signatures associated with stable colony morphology and with unstable colony morphology, although the typical pluripotency markers (POU5F1, SOX2) were present with both morphologies. Notably, epithelial to mesenchymal transition (EMT) protein markers were associated with unstable colony morphology, and the transforming growth factor beta (TGFB) signalling pathway was predicted as one of the main regulator pathways involved in this process. Furthermore, we identified specific proteins that separated the stable from the unstable state. Finally, we assessed both spontaneous embryonic body (EB) formation and directed differentiation and showed that reprogrammed lines with an unstable colony morphology had reduced differentiation capacity. To conclude, we found that different defined patterns of colony morphology in reprogrammed cells were associated with distinct proteomic profiles and different outcomes in differentiation capacity.publishedVersio

Proliferation and recapitulation of developmental patterning associated with regulative regeneration of the spinal cord neural tube.

Developmental patterning during regulative regeneration of the chicken embryo spinal neural tube was characterized by assessing proliferation and the expression of transcription factors specific to neural progenitor and postmitotic neuron populations. One to several segments of the thoracolumbar neural tube were selectively excised unilaterally to initiate regeneration. The capacity for regeneration depended on the stage when ablation was performed and the extent of tissue removed. 20% of surviving embryos exhibited complete regulative regeneration, wherein the missing hemi-neural tube was reconstituted to normal size and morphology. Fate-mapping of proliferative adjacent tissue indicated contributions from the opposite side of the neural tube and potentially from the ipsilateral neural tube rostral and caudal to the lesion. Application of the thymidine analog EdU (5-ethynyl-2'-deoxyuridine) demonstrated a moderate increase in cell proliferation in lesioned relative to control embryos, and quantitative PCR demonstrated a parallel moderate increase in transcription of proliferation-related genes. Mathematical calculation showed that such modest increases are sufficient to account for the amount of regenerated tissue. Within the regenerated neural tube the expression pattern of progenitor-specific transcription factors was recapitulated in the separate advancing ventral and dorsal fronts of regeneration, with no evidence of abnormal mixing of progenitor subpopulations, indicating that graded patterning mechanisms do not require continuity of neural tube tissue along the dorsoventral axis and do not involve a sorting out of committed progenitors. Upon completion of the regeneration process, the pattern of neuron-specific transcription factor expression was essentially normal. Modest deficits in the numbers of transcription factor-defined neuron types were evident in the regenerated tissue, increasing particularly in dorsal neuron types with later lesions. These results confirm the regulative potential of the spinal neural tube and demonstrate a capacity for re-establishing appropriate cellular patterning despite a grossly abnormal morphogenetic situation.Norwegian Research Council Norwegian Center for Stem Cell Research Norway-Hungary Cultural Exchange Progra

Novel methods to assess environmental, economic, and social sustainability of main agricultural regions in China

<div><p>Teleosts show a great variety in visual opsin complement, due to both gene duplication and gene loss. The repertoire ranges from one subfamily of visual opsins (scotopic vision) including rod opsin only retinas seen in many deep-sea species to multiple subfamilies of visual opsins in some pelagic species. We have investigated the opsin repertoire of Atlantic cod (<i>Gadus morhua</i>) using information in the recently sequenced cod genome and found that despite cod not being a deep sea species it lacks visual subfamilies sensitive towards the most extreme parts of the light spectra representing UV and red light. Furthermore, we find that Atlantic cod has duplicated paralogs of both blue-sensitive SWS2 and green-sensitive RH2 subfamilies, with members belonging to each subfamily linked in tandem within the genome (two SWS2-, and three RH2A genes, respectively). The presence of multiple cone opsin genes indicates that there have been duplication events in the cod ancestor SWS2 and RH2 opsins producing paralogs that have been retained in Atlantic. Our results are supported by expressional analysis of cone opsins, which further revealed an ontogenetic change in the array of cone opsins expressed. These findings suggest life stage specific programs for opsin regulation which could be linked to habitat changes and available light as the larvae is transformed into an early juvenile. Altogether we provide the first molecular evidence for color vision driven by only two families of cone opsins due to gene loss in a teleost.</p></div

Topographic mRNA expression of visual opsins in cod larvae.

<p>The retinal expression of different cone opsins were investigated by <i>in situ</i> hybridization on 18 days post fertilized cod larvae (2 days post hatching). The pictures in the left row (A, D, G, J, and M) show dorsal view of whole mount in situ on larvae, while the centre row of pictures (B, E, H, K and N) show whole mount of a right eye, lateral view; with larval anterior to the left. Sectional in situ is shown by sagittal sections in the left row of pictures (C, F, I, L and O). Expression of the various cone opsins was visualized by specific dig-labelled RNA probes: <i>SWS2A</i> (A, B and C), <i>SWS2B</i> (D, E and F), <i>RH2A-1</i> (G, H and I), <i>RH2A-2</i> (J, K and L) and <i>RH2A-3</i> (M, N and O). Arrows indicate location of choroid fissure. Scale bars, 500 µm.</p

Synteny of cod cone opsins.

<p>(A) The scaffold ATLCOD1Cs2467 was found to contain the <i>SWS2-LWS</i> opsin syntenic region conserved within teleosts. The position of the <i>SWS2</i> genes and the phylogenetic analysis indicates that cod displays the same picture as seen in medaka with the <i>SWS2</i> genes being <i>SWS2A</i> and –<i>B</i>. The <i>SWS</i>* is an incomplete pseudogene. The genome lacks the <i>LWS</i> gene(s) and the region between the cod <i>SWS2B</i> and the <i>GNL3L</i> in cod is only 4.5 kb. (B) The scaffold ATLCOD1Cs169 was found to contain the <i>RH2A</i> opsin syntenic region. The number and orientation of the <i>RH2A</i> genes differs between the species, but the surrounding genes are the same except for cod which is linked to different genes upstream of the <i>RH2A</i> genes. The distance between the green opsins and the non-visual parapinopsin in cod is approximately 180 kb. (C) The scaffold ATLCOD1Bc1499404 was found to contain the <i>SWS1</i> opsin syntenic region. The closest genes surrounding the <i>SWS1</i> gene in zebrafish are also clustered in cod, but the <i>SWS1</i> gene is missing. The genes downstream of <i>SWS1</i> are different in medaka and stickleback.</p

Reprogrammed Cells Display Distinct Proteomic SignaturesAssociated with Colony Morphology Variability

Human induced pluripotent stem cells (hiPSCs) are of high interest because they can be differentiated into a vast range of different cell types. Ideally, reprogrammed cells should sustain long-term culturing in an undifferentiated state. However, some reprogrammed cell lines represent an unstable state by spontaneously differentiating and changing their cellular phenotype and colony morphology. This phenomenon is not fully understood, and no method is available to predict it reliably. In this study, we analyzed and compared the proteome landscape of 20 reprogrammed cell lines classified as stable and unstable based on long-term colony morphology. We identified distinct proteomic signatures associated with stable colony morphology and with unstable colony morphology, although the typical pluripotency markers (POU5F1, SOX2) were present with both morphologies. Notably, epithelial to mesenchymal transition (EMT) protein markers were associated with unstable colony morphology, and the transforming growth factor beta (TGFB) signalling pathway was predicted as one of the main regulator pathways involved in this process. Furthermore, we identified specific proteins that separated the stable from the unstable state. Finally, we assessed both spontaneous embryonic body (EB) formation and directed differentiation and showed that reprogrammed lines with an unstable colony morphology had reduced differentiation capacity. To conclude, we found that different defined patterns of colony morphology in reprogrammed cells were associated with distinct proteomic profiles and different outcomes in differentiation capacity

Cone opsins expressed in adult cod.

<p><i>In situ</i> hybridization shows that adult cod express both subtypes of <i>SWS2</i>; <i>SWS2A</i> and <i>SWS2B</i>, while only one RH2A subtype, the <i>RH2A-1</i> is expressed in the retina. The <i>SWS2A</i> opsin (A and D) seems to be expressed in a higher number of transcripts in each photoreceptor cell compared with the <i>SWS2B</i> opsin (B and E). Both <i>SWS2A</i> and <i>SWS2B</i> expressing photoreceptors are found at some distance apart, and appear to form a regular pattern. The green RH2A-1 opsin (C and F) dominates in the retina of adult cod with only a few cells lacking expression. Scale bar, 20 µm.</p

Phylogeny of green-sensitive RH2 opsins in teleosts.

<p>Coding nucleotide sequences were aligned with Clustal W, and the tree was generated by maximum likelihood method. The bootstrap confidence value (1000 replicates) is shown for each branch, and the lamprey (<i>G. australis</i>) sequence (accession number AY366494) was used as outgroup. The scale bar is equal to 0.05 substitutions per site. Cod RH2A genes are highlighted within the green box. The sequences used for generating the tree are: Atlantic cod (<i>G. morhua</i>), AF385824, KJ572530 and KJ572531; Malawi cichlid (<i>M. pyrsonotos</i>), ADI77346 and ADI72269; tilapia (O. niloticus), ADW80524, ADW80525 and ADW80526; guppy (<i>P. reticulata</i>), ABB69697 and ABB69696; halibut (<i>H. hippoglossus</i>), AAM17916; stickleback (<i>G. aculeatus</i>), AGL76515; fugu (<i>T. rupri</i>), AAF44648; salmon (<i>S. salar</i>), AAP58323, trout (<i>O. mykiss</i>), AAP35093; ayu (<i>P. altivelis</i>), BAD54744 and BAD54745; goldfish (<i>C. auratus</i>), AAA49168 and AAA49169; zebrafish (<i>D. rario</i>), AAD24752, AAD24753, BAC24131 and BAC24130; American chameleon (<i>A. carolinensis</i>), AH007735; chicken (<i>G. gallus</i>), NM_205490 and coelacanth (<i>L. chalumnae</i>), AAD30520.</p

Phylogeny of blue-sensitive SWS2 opsins in teleosts.

<p>Deduced amino acid sequences were aligned with Clustal W, and the tree was generated by maximum likelihood method. The bootstrap confidence value (1000 replicates) is shown for each branch, and the lamprey (<i>G. australis</i>) SWS1 (accession number: AAR14684) sequence was used as outgroup. The scale bar is equal to 0.1 substitutions per site. Cod SWS2 genes are highlighted in blue. The protein sequences used for generating the tree are: Cod (<i>G. morhua</i>), Q5K6I6 and KJ572531 (translated); Malawi cichlid (<i>M. lateristriga</i>), Q4VPY3 and Q4VPX4; tilapia (<i>O. niloticus</i>), Q9I9I7 and Q9I9I9; salmon (<i>S. salar</i>), Q6XR07; trout (<i>O. mykiss</i>), Q7ZT59; Takifugu (<i>T. rupri</i>), Q6J5J9; zebrafish (<i>D. rario</i>), Q9W6A8; goldfish (<i>C. auratus</i>), P32310; guppy (<i>P. reticulata</i>), Q0H3C3; European eel (<i>A. anguilla</i>), ACT34385; chicken (<i>G. gallus</i>), NP_990848; tiger salamander (<i>A. tigrinum</i>), AAC96069 and spotted green pufferfish (<i>T. nigroviridis</i>), Q6J5J8.</p