NEAT1 polyA-modulating antisense oligonucleotides reveal opposing functions for both long non-coding RNA isoforms in neuroblastoma.

Many long non-coding RNAs (lncRNA) are highly dysregulated in cancer and are emerging as therapeutic targets. One example is NEAT1, which consists of two overlapping lncRNA isoforms, NEAT1_1 (3.7 kb) and NEAT1_2 (23 kb), that are functionally distinct. The longer NEAT1_2 is responsible for scaffolding gene-regulatory nuclear bodies termed paraspeckles, whereas NEAT1_1 is involved in paraspeckle-independent function. The NEAT1 isoform ratio is dependent on the efficient cleavage and polyadenylation of NEAT1_1 at the expense of NEAT1_2. Here, we developed a targeted antisense oligonucleotide (ASO) approach to sterically block NEAT1_1 polyadenylation processing, achieving upregulation of NEAT1_2 and abundant paraspeckles. We have applied these ASOs to cells of the heterogeneous infant cancer, neuroblastoma, as we found higher NEAT1_1:NEAT1_2 ratio and lack of paraspeckles in high-risk neuroblastoma cells. These ASOs decrease NEAT1_1 levels, increase NEAT1_2/paraspeckles and concomitantly reduce cell viability in high-risk neuroblastoma specifically. In contrast, overexpression of NEAT1_1 has the opposite effect, increasing cell proliferation. Transcriptomic analyses of high-risk neuroblastoma cells with altered NEAT1 ratios and increased paraspeckle abundance after ASO treatment showed an upregulation of differentiation pathways, as opposed to the usual aggressive neuroblastic phenotype. Thus, we have developed potential anti-cancer ASO drugs that can transiently increase growth-inhibiting NEAT1_2 RNA at the expense of growth-promoting NEAT1_1 RNA. These ASOs, unlike others that degrade lncRNAs, provide insights into the importance of altering lncRNA polyadenylation events to suppress tumorigenesis as a strategy to combat cancer.

Structure illumination microscopy imaging of lipid vesicles in live bacteria with naphthalimide-appended organometallic complexes.

There is a lack of molecular probes for imaging bacteria, in comparison to the array of such tools available for the imaging of mammalian cells. Here, organometallic molecular probes have been developed and assessed for bacterial imaging, designed to have the potential to support multiple imaging modalities. The chemical structure of the probes is designed around a metal-naphthalimide structure. The 4-amino-1,8-naphthalimide moiety, covalently appended through a pyridine ancillary ligand, acts as a luminescent probe for super-resolution microscopy. On the other hand, the metal centre, rhenium(i) or platinum(ii) in the current study, enables techniques such as nanoSIMS. While the rhenium(i) complex was not sufficiently stable to be used as a probe, the platinum(ii) analogue showed good chemical and biological stability. Structured illumination microscopy (SIM) imaging on live Bacillus cereus confirmed the suitability of the probe for super-resolution microscopy. NanoSIMS analysis was used to monitor the uptake of the platinum(ii) complex within the bacteria and demonstrate the potential of this chemical architecture to enable multimodal imaging. The successful combination of these two moieties introduces a platform that could lead to a versatile range of multi-functional probes for bacteria.

Complementary Approaches to Imaging Subcellular Lipid Architectures in Live Bacteria Using Phosphorescent Iridium Complexes and Raman Spectroscopy.

A family of three neutral iridium(III) tetrazolato complexes are investigated as bacterial imaging agents. The complexes offer a facile tuning of the emission colour from green (520 nm) to red (600 nm) in aqueous media, while keeping the excitation wavelength unchanged. The three complexes do not inhibit the bacterial growth of Bacillus Cereus, used as a model in this study, and exhibit extremely fast cellular uptake. After a minute incubation time, the nontoxic complexes show subcellular localisation in spherical structures identified as lipid vacuoles. Confocal Raman imaging has been exploited for the first time on live bacteria, to provide direct and label-free mapping of the lipid-enriched organelles within B. cereus, complementing the use of luminescent probes. Examination of the Raman spectra not only confirmed the presence of lipophilic inclusions in B. cereus but offered additional information about their chemical composition, suggesting that the lipid vacuoles may contain polyhydroxybutyrate (PHB).

Distinction Between Active and Passive Targeting of Nanoparticles Dictate Their Overall Therapeutic Efficacy.

The role of nanoparticles in cancer medicine is vast with debate still surrounding the distinction between therapeutic efficacy of actively targeted nanoparticles versus passively targeted systems for drug delivery. While it is commonly accepted that methodologies that result in homing a high concentration of drug loaded nanoparticles to the tumor is beneficial, the role of intracellular trafficking of these nanoparticles in dictating the overall therapeutic outcome remains unresolved. Herein we demonstrate that the therapeutic outcome of drug loaded nanoparticles is governed beyond simply enabling nanoparticle internalization in cells. Using two model polymeric nanoparticles, one decorated with the GE11 peptide for active targeting of the epidermal growth factor receptor (EGFR) and the other without, we demonstrate that EGFR mediated intracellular internalization results in an enhanced therapeutic effect compared to the nontargeted formulation. Our findings demonstrate that the intracellular destination of nanoparticles beyond its ability to internalize is an important parameter that has to be accounted for in the design of targeted drug delivery systems.

Molecular exploration of fossil eggshell uncovers hidden lineage of giant extinct bird.

The systematics of Madagascar's extinct elephant birds remains controversial due to large gaps in the fossil record and poor biomolecular preservation of skeletal specimens. Here, a molecular analysis of 1000-year-old fossil eggshells provides the first description of elephant bird phylogeography and offers insight into the ecology and evolution of these flightless giants. Mitochondrial genomes from across Madagascar reveal genetic variation that is correlated with eggshell morphology, stable isotope composition, and geographic distribution. The elephant bird crown is dated to ca. 30 Mya, when Madagascar is estimated to have become less arid as it moved northward. High levels of between-clade genetic variation support reclassifying Mullerornis into a separate family. Low levels of within-clade genetic variation suggest there were only two elephant bird genera existing in southern Madagascar during the Holocene. However, we find an eggshell collection from Madagascar's far north that represents a unique lineage of Aepyornis. Furthermore, divergence within Aepyornis coincides with the aridification of Madagascar during the early Pleistocene ca. 1.5 Ma, and is consistent with the fragmentation of populations in the highlands driving diversification and the evolution of extreme gigantism over shorts timescales. We advocate for a revision of their taxonomy that integrates palaeogenomic and palaeoecological perspectives.

Distinct differences between calvarial and long bone osteocytes in cell morphologies, gene expression and aging responses.

Osteocytes are the terminally differentiated bone cells resulted from bone formation. Although there are two distinct processes of bone formation, intramembranous and endochondral ossifications contributing to the formation of calvarial and long bones, it is not clear whether the distinct pathways determine the differences between calvaria and femoral cortical bone derived osteocytes. In the present study, we employed confocal structured illumination microscopy and mRNA-sequencing analysis to characterize the morphologic and transcriptomic expression of osteocytes from murine calvaria and mid-shaft femoral cortical bone. Structured illumination microscopy and geometric modelling showed round shaped and irregularly scattered calvarial osteocytes compared to spindle shaped and orderly arrayed cortical osteocytes. mRNA-sequencing analysis indicated different transcriptomic profiles between calvarial and cortical osteocytes and provided evidence that mechanical response of osteocytes may contribute to geometrical differences. Furthermore, transcriptomic analysis showed that these two groups of osteocytes come from distinct pathways with 121 ossification-related genes differentially expressed. Analysis of correlation between ossification and osteocyte geometries via a Venn diagram showed that several genes related to ossification, cytoskeleton organization and dendrite development were differentially expressed between calvarial and cortical osteocytes. Finally, we demonstrated that aging disrupted the organization of dendrites and cortical osteocytes but had no significant effects on calvarial osteocytes. Together, we conclude that calvarial and cortical osteocytes are different in various aspects, which is probably the consequence of their distinct pathways of ossification.

Single Stranded Fully Modified-Phosphorothioate Oligonucleotides can Induce Structured Nuclear Inclusions, Alter Nuclear Protein Localization and Disturb the Transcriptome In Vitro.

Oligonucleotides and nucleic acid analogues that alter gene expression are now showing therapeutic promise in human disease. Whilst the modification of synthetic nucleic acids to protect against nuclease degradation and to influence drug function is common practice, such modifications may also confer unexpected physicochemical and biological properties. Gapmer mixed-modified and DNA oligonucleotides on a phosphorothioate backbone can bind non-specifically to intracellular proteins to form a variety of toxic inclusions, driven by the phosphorothioate linkages, but also influenced by the oligonucleotide sequence. Recently, the non-antisense or other off-target effects of 2' O- fully modified phosphorothioate linkage oligonucleotides are becoming better understood. Here, we report chemistry-specific effects of oligonucleotides composed of modified or unmodified bases, with phosphorothioate linkages, on subnuclear organelles and show altered distribution of nuclear proteins, the appearance of highly stable and strikingly structured nuclear inclusions, and disturbed RNA processing in primary human fibroblasts and other cultured cells. Phosphodiester, phosphorodiamidate morpholino oligomers, and annealed complimentary phosphorothioate oligomer duplexes elicited no such consequences. Disruption of subnuclear structures and proteins elicit severe phenotypic disturbances, revealed by transcriptomic analysis of transfected fibroblasts exhibiting such disruption. Our data add to the growing body of evidence of off-target effects of some phosphorothioate nucleic acid drugs in primary cells and suggest alternative approaches to mitigate these effects.

Multimodal Imaging and Analysis of the Neuroanatomical Organization of the Primary Olfactory Inputs in the Brownbanded Bamboo Shark, Chiloscyllium punctatum.

There is currently a limited understanding of the morphological and functional organization of the olfactory system in cartilaginous fishes, particularly when compared to bony fishes and terrestrial vertebrates. In this fish group, there is a clear paucity of information on the characterization, density, and distribution of olfactory receptor neurons (ORNs) within the sensory olfactory epithelium lining the paired olfactory rosettes, and their functional implications with respect to the hydrodynamics of incurrent water flow into the nares. This imaging study examines the brownbanded bamboo shark Chiloscyllium punctatum (Elasmobranchii) and combines immunohistochemical labeling using antisera raised against five G-protein α-subunits (Gαs/olf, Gαq/ 11 / 14, Gαi- 1 / 2 / 3, Gαi- 3, Gα o ) with light and electron microscopy, to characterize the morphological ORN types present. Three main ORNs ("long", "microvillous" and "crypt-like") are confirmed and up to three additional microvilli-bearing types are also described; "Kappe-like" (potential or homologous "Kappe" as in teleosts), "pear-shaped" and "teardrop-shaped" cells. These morphotypes will need to be confirmed molecularly in the future. Using X-ray diffusible iodine-based contrast-enhanced computed tomography (diceCT), high-resolution scans of the olfactory rosettes, olfactory bulbs (OBs), peduncles, and telencephalon reveal a lateral segregation of primary olfactory inputs within the OBs, with distinct medial and lateral clusters of glomeruli, suggesting a potential somatotopic organization. However, most ORN morphotypes are found to be ubiquitously distributed within the medial and lateral regions of the olfactory rosette, with at least three microvilli-bearing ORNs labeled with anti-Gα o found in significantly higher densities in lateral lamellae [in lateral lamellae] and on the anterior portion of lamellae (facing the olfactory cavity). These microvilli-bearing ORN morphotypes (microvillous, "Kappe-like," "pear-shaped," and "teardrop-shaped") are the most abundant across the olfactory rosette of this species, while ciliated ORNs are less common and crypt cells are rare. Spatial simulations of the fluid dynamics of the incurrent water flow into the nares and within the olfactory cavities indicate that the high densities of microvilli-bearing ORNs located within the lateral region of the rosette are important for sampling incoming odorants during swimming and may determine subsequent tracking behavior.