circPVT1 and PVT1/AKT3 show a role in cell proliferation, apoptosis, and tumor subtype-definition in small cell lung cancer.

Small cell lung cancer (SCLC) is treated as a homogeneous disease, although the expression of NEUROD1, ASCL1, POU2F3, and YAP1 identifies distinct molecular subtypes. The MYC oncogene, amplified in SCLC, was recently shown to act as a lineage-specific factor to associate subtypes with histological classes. Indeed, MYC-driven SCLCs show a distinct metabolic profile and drug sensitivity. To disentangle their molecular features, we focused on the co-amplified PVT1, frequently overexpressed and originating circular (circRNA) and chimeric RNAs. We analyzed hsa_circ_0001821 (circPVT1) and PVT1/AKT3 (chimPVT1) as examples of such transcripts, respectively, to unveil their tumorigenic contribution to SCLC. In detail, circPVT1 activated a pro-proliferative and anti-apoptotic program when over-expressed in lung cells, and knockdown of chimPVT1 induced a decrease in cell growth and an increase of apoptosis in SCLC in vitro. Moreover, the investigated PVT1 transcripts underlined a functional connection between MYC and YAP1/POU2F3, suggesting that they contribute to the transcriptional landscape associated with MYC amplification. In conclusion, we have uncovered a functional role of circular and chimeric PVT1 transcripts in SCLC; these entities may prove useful as novel biomarkers in MYC-amplified tumors.

Genome characterization and CRISPR-Cas9 editing of a human neocentromere.

The maintenance of genome integrity is ensured by proper chromosome inheritance during mitotic and meiotic cell divisions. The chromosomal counterpart responsible for chromosome segregation to daughter cells is the centromere, at which the spindle apparatus attaches through the kinetochore. Although all mammalian centromeres are primarily composed of megabase-long repetitive sequences, satellite-free human neocentromeres have been described. Neocentromeres and evolutionary new centromeres have revolutionized traditional knowledge about centromeres. Over the past 20 years, insights have been gained into their organization, but in spite of these advancements, the mechanisms underlying their formation and evolution are still unclear. Today, through modern and increasingly accessible genome editing and long-read sequencing techniques, research in this area is undergoing a sudden acceleration. In this article, we describe the primary sequence of a previously described human chromosome 3 neocentromere and observe its possible evolution and repair results after a chromosome breakage induced through CRISPR-Cas9 technologies. Our data represent an exciting advancement in the field of centromere/neocentromere evolution and chromosome stability.

A single amino acid switch converts the Sleeping Beauty transposase into an efficient unidirectional excisionase with utility in stem cell reprogramming.

The Sleeping Beauty (SB) transposon is an advanced tool for genetic engineering and a useful model to investigate cut-and-paste DNA transposition in vertebrate cells. Here, we identify novel SB transposase mutants that display efficient and canonical excision but practically unmeasurable genomic re-integration. Based on phylogenetic analyses, we establish compensating amino acid replacements that fully rescue the integration defect of these mutants, suggesting epistasis between these amino acid residues. We further show that the transposons excised by the exc+/int- transposase mutants form extrachromosomal circles that cannot undergo a further round of transposition, thereby representing dead-end products of the excision reaction. Finally, we demonstrate the utility of the exc+/int- transposase in cassette removal for the generation of reprogramming factor-free induced pluripotent stem cells. Lack of genomic integration and formation of transposon circles following excision is reminiscent of signal sequence removal during V(D)J recombination, and implies that cut-and-paste DNA transposition can be converted to a unidirectional process by a single amino acid change.

Functional Characterization of the N-Terminal Disordered Region of the piggyBac Transposase.

The piggyBac DNA transposon is an active element initially isolated from the cabbage looper moth, but members of this superfamily are also present in most eukaryotic evolutionary lineages. The functionally important regions of the transposase are well described. There is an RNase H-like fold containing the DDD motif responsible for the catalytic DNA cleavage and joining reactions and a C-terminal cysteine-rich domain important for interaction with the transposon DNA. However, the protein also contains a ~100 amino acid long N-terminal disordered region (NTDR) whose function is currently unknown. Here we show that deletion of the NTDR significantly impairs piggyBac transposition, although the extent of decrease is strongly cell-type specific. Moreover, replacing the NTDR with scrambled but similarly disordered sequences did not rescue transposase activity, indicating the importance of sequence conservation. Cell-based transposon excision and integration assays reveal that the excision step is more severely affected by NTDR deletion. Finally, bioinformatic analyses indicated that the NTDR is specific for the piggyBac superfamily and is also present in domesticated, transposase-derived proteins incapable of catalyzing transposition. Our results indicate an essential role of the NTDR in the "fine-tuning" of transposition and its significance in the functions of piggyBac-originated co-opted genes.

A genomic survey of Tc1-mariner transposons in nematodes suggests extensive horizontal transposon transfer events.

The number of reports concerning horizontal transposon transfers (HTT) in metazoan species is considerably increased, alongside with the exponential growth of genomic sequence data However, our understanding of the mechanisms of such phenomenon is still at an early stage. Nematodes constitute an animal phylum successfully adapted to almost every ecosystem and for this reason could potentially contribute to spreading the genetic information through horizontal transfer. To date, few studies describe HTT of nematode retrotransposons. This is due to the lack of annotation of transposable elements in the sequenced nematode genomes, especially DNA transposons, which are acknowledged as the best horizontal travelers among mobile sequences. We have therefore started a survey of DNA transposons and their possible involvement in HTT in sequenced nematode genomes. Here, we describe 83 new Tc1/mariner elements distributed in 17 nematode species. Among them, nine families were possibly horizontally transferred between nematodes and the most diverse animal species, including ants as preferred partner of HTT. The results obtained suggest that HTT events involving nematodes Tc1/mariner elements are not uncommon, and that nematodes could have a possible role as transposon reservoir that, in turn, can be redistributed among animal genomes. Overall, this could be relevant to understand how the inter-species genetic flows shape the landscape of genetic variation of organisms inhabiting specific environmental communities.

Nonstandard Neutrino Interactions as a Solution to the NOνA and T2K Discrepancy.

The latest data of the two long-baseline accelerator experiments NOνA and T2K, interpreted in the standard three-flavor scenario, display a discrepancy. A mismatch in the determination of the standard CP phase δ_{CP} extracted by the two experiments is evident in the normal neutrino mass ordering. While NOνA prefers values close to δ_{CP}∼0.8π, T2K identifies values of δ_{CP}∼1.4π. Such two estimates are in disagreement at more than 90% C.L. for 2 degrees of freedom. We show that such a tension can be resolved if one hypothesizes the existence of complex neutral-current nonstandard interactions (NSIs) of the flavor changing type involving the e-µ or the e-τ sectors with couplings |ϵ_{eµ}|∼|ϵ_{eτ}|∼0.2. Remarkably, in the presence of such NSIs, both experiments point towards the same common value of the standard CP phase δ_{CP}∼3π/2. Our analysis also highlights an intriguing preference for maximal CP violation in the nonstandard sector with the NSI CP phases having best fit close to ϕ_{eµ}∼ϕ_{eτ}∼3π/2, hence pointing towards imaginary NSI couplings.

Transposable elements: a jump toward the future of expression vectors.

Expression vectors (EVs) are artificial nucleic acid molecules with a modular structure that allows for the transcription of DNA sequences of interest in either cellular or cell-free environments. These vectors have emerged as cross-disciplinary tools with multiple applications in an expanding Life Sciences market. The cis-regulatory sequences (CRSs) that control the transcription in EVs are typically sourced from either viruses or from characterized genes. However, the recent advancement in transposable elements (TEs) technology provides attractive alternatives that may enable a significant improvement in the design of EVs. Commonly known as "jumping genes," due to their ability to move between genetic loci, TEs are constitutive components of both eukaryotic and prokaryotic genomes. TEs harbor native CRSs that allow the regulated transcription of transposition-related genes. However, some TE-related CRSs display striking characteristics, which provides the opportunity to reconsider TEs as lead actors in the design of EVs. In this article, we provide a synopsis of the transcriptional control elements commonly found in EVs together with an extensive discussion of their advantages and limitations. We also highlight the latest findings that may allow for the implementation of TE-derived sequences in the EVs feasible, possibly improving existing vectors. By introducing this new concept of TEs as a source of regulatory sequences, we aim to stimulate a profitable discussion of the potential advantages and benefits of developing a new generation of EVs based on the use of TE-derived control sequences.

Long Non-Coding RNA Landscape in Prostate Cancer Molecular Subtypes: A Feature Selection Approach.

Prostate cancer is one of the most common malignancies in men. It is characterized by a high molecular genomic heterogeneity and, thus, molecular subtypes, that, to date, have not been used in clinical practice. In the present paper, we aimed to better stratify prostate cancer patients through the selection of robust long non-coding RNAs. To fulfill the purpose of the study, a bioinformatic approach focused on feature selection applied to a TCGA dataset was used. In such a way, LINC00668 and long non-coding(lnc)-SAYSD1-1, able to discriminate ERG/not-ERG subtypes, were demonstrated to be positive prognostic biomarkers in ERG-positive patients. Furthermore, we performed a comparison between mutated prostate cancer, identified as "classified", and a group of patients with no peculiar genomic alteration, named "not-classified". Moreover, LINC00920 lncRNA overexpression has been linked to a better outcome of the hormone regimen. Through the feature selection approach, it was found that the overexpression of lnc-ZMAT3-3 is related to low-grade patients, and three lncRNAs: lnc-SNX10-87, lnc-AP1S2-2, and ADPGK-AS1 showed, through a co-expression analysis, significant correlation values with potentially druggable pathways. In conclusion, the data mining of publicly available data and robust bioinformatic analyses are able to explore the unknown biology of malignancies.

Neutrino Mass Ordering Obscured by Nonstandard Interactions.

One of the major open questions in particle physics is the issue of the neutrino mass ordering (NMO). The current data of the two long-baseline experiments NOνA and T2K, interpreted in the standard three-flavor scenario, provide a ∼2.4σ indication in favor of the normal neutrino mass ordering. We show that such an indication is completely washed out if one assumes the existence of neutral-current nonstandard interactions (NSI) of the flavor changing type involving the e-τ flavors. This implies that the claim for a discovery of the NMO will require a careful consideration of the impact of hypothetical NSI.

Epigenetically induced ectopic expression of UNCX impairs the proliferation and differentiation of myeloid cells.

We here describe a leukemogenic role of the homeobox gene UNCX, activated by epigenetic modifications in acute myeloid leukemia (AML). We found the ectopic activation of UNCX in a leukemia patient harboring a t(7;10)(p22;p14) translocation, in 22 of 61 of additional cases [a total of 23 positive patients out of 62 (37.1%)], and in 6 of 75 (8%) of AML cell lines. UNCX is embedded within a low-methylation region (canyon) and encodes for a transcription factor involved in somitogenesis and neurogenesis, with specific expression in the eye, brain, and kidney. UNCX expression turned out to be associated, and significantly correlated, with DNA methylation increase at its canyon borders based on data in our patients and in archived data of patients from The Cancer Genome Atlas. UNCX-positive and -negative patients displayed significant differences in their gene expression profiles. An enrichment of genes involved in cell proliferation and differentiation, such as MAP2K1 and CCNA1, was revealed. Similar results were obtained in UNCX-transduced CD34+ cells, associated with low proliferation and differentiation arrest. Accordingly, we showed that UNCX expression characterizes leukemia cells at their early stage of differentiation, mainly M2 and M3 subtypes carrying wild-type NPM1 We also observed that UNCX expression significantly associates with an increased frequency of acute promyelocytic leukemia with PML-RARA and AML with t(8;21)(q22;q22.1); RUNX1-RUNX1T1 classes, according to the World Health Organization disease classification. In summary, our findings suggest a novel leukemogenic role of UNCX, associated with epigenetic modifications and with impaired cell proliferation and differentiation in AML.

Octant of θ_{23} in Danger with a Light Sterile Neutrino.

Present global fits of world neutrino data hint towards nonmaximal θ_{23} with two nearly degenerate solutions, one in the lower octant (θ_{23}<π/4), and the other in the higher octant (θ_{23}>π/4). This octant ambiguity of θ_{23} is one of the fundamental issues in the neutrino sector, and its resolution is a crucial goal of next-generation long-baseline (LBL) experiments. In this Letter, we address, for the first time, the impact of a light eV-scale sterile neutrino towards such a measurement, taking the Deep Underground Neutrino Experiment as a case study. In the so-called 3+1 scheme involving three active and one sterile neutrinos, the ν_{µ}→ν_{e} transition probability probed in the LBL experiments acquires a new interference term via active-sterile oscillations. We find that this interference term can mimic a swap of the θ_{23} octant, even if one uses the information from both neutrino and antineutrino channels. As a consequence, the sensitivity to the octant of θ_{23} can be completely lost, and this may have serious implications for our understanding of neutrinos from both the experimental and theoretical perspectives.

Role of somatomedin-B-like domains on ENPP1 inhibition of insulin signaling.

The exact mechanism by which ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) inhibits insulin signaling is not known. ENPP1 contains two somatomedin-B-like domains (i.e. SMB 1 and 2) involved in ENPP1 dimerization in animal cells. The aim of the present study was to investigate if these domains modulate ENPP1 inhibitory activity on insulin signaling in human insulin target cells (HepG2). ENPP1 (ENPP1-3'myc), ENPP1 deleted of SMB 1 (ENPP1-ΔI-3'myc) or of SMB 2 (ENPP1-ΔII-3'myc) domain were cloned in frame with myc tag in mammalian expression vector pRK5. Plasmids were transiently transfected in human liver HepG2 cells. ENPP1 inhibitory activity on insulin signaling, dimerization and protein-protein interaction with insulin receptor (IR), reported to mediate the modulation of ENPP1 inhibitory activity, were studied. As compared to untransfected cells, a progressive increase of ENPP1 inhibitory activity on insulin-induced IR ß-subunit autophosphorylation and on Akt-S(473) phosphorylation was observed in ENPP1-3'myc, ENPP1-ΔI-3'myc and ENPP1-ΔII-3'myc cells. Under non reducing conditions a 260 kDa homodimer, indicating ENPP1 dimerization, was observed. The ratio of non reduced (260 kDa) to reduced (130 kDa) ENPP1 was significantly decreased by two thirds in ENPP1-ΔII-3'myc vs. ENPP1-3'myc but not in ENPP1-ΔI-3'myc. A similar ENPP1/IR interaction was detectable by co-immunoprecipitation in ENPP1-3'myc, ENPP1-ΔI-3'myc and ENPP1-ΔII-3'myc cells. In conclusion, SMB 1 and SMB 2 are negative modulators of ENPP1 inhibitory activity on insulin signaling. For SMB 2 such effect might be mediated by a positive role on protein dimerization.

Flying toward a plastic-free world: Can Drosophila serve as a model organism to develop new strategies of plastic waste management?

The last century was dominated by the widespread use of plastics, both in terms of invention and increased usage. The environmental challenge we currently face is not just about reducing plastic usage but finding new ways to manage plastic waste. Recycling is growing but remains a small part of the solution. There is increasing focus on studying organisms and processes that can break down plastics, offering a modern approach to addressing the environmental crisis. Here, we provide an overview of the organisms associated with plastics biodegradation, and we explore the potential of harnessing and integrating their genetic and biochemical features into a single organism, such as Drosophila melanogaster. The remarkable genetic engineering and microbiota manipulation tools available for this organism suggest that multiple features could be amalgamated and modeled in the fruit fly. We outline feasible genetic engineering and gut microbiome engraftment strategies to develop a new class of plastic-degrading organisms and discuss of both the potential benefits and the limitations of developing such engineered Drosophila melanogaster strains.

Pheromone-mediated command from the female to male clock induces and synchronizes circadian rhythms of the moth Spodoptera littoralis.

To extract any adaptive benefit, the circadian clock needs to be synchronized to the 24-h day-night cycles. We have investigated if it is a general property of the brain's circadian clock to recognize social interactions as external time givers. Sociosexual interactions with the opposite sex are universal, prevalent even in the lives of solitary animals. The solitary adult life of the Spodoptera littoralis moth is singularly dedicated to sex, offering an ideal context for exploring the impact of sociosexual cues on circadian timekeeping. We have identified specific olfactory cues responsible for social entrainment, revealing a surprisingly strong influence of pheromone-mediated remote sociosexual interactions on circadian rhythms. Males' free-running rhythms are induced and synchronized by the sex pheromone that the female releases in a rhythmic fashion, highlighting a hierarchical relation between the female and male circadian oscillators. Even a single pulse of the sex pheromone altered clock gene expression in the male brain, surpassing the effect of light on the clock. Our finding of a daytime-dependent, lasting impact of pheromone on male's courtship efficacy indicates that circadian timing in moths is a trait under sexual selection. We have identified specific components of the sex-pheromone blend that lack mate-attractive property but have powerful circadian effects, providing rationale for their continued retention by the female. We show that such volatiles, when shared across sympatric moth species, can trigger communal synchronization. Our results suggest that the sex pheromone released by female moths entrains males' behavioral activity rhythm to ensure synchronized timing of mating.

Structural and Comparative Analyses of Insects Suggest the Presence of an Ultra-Conserved Regulatory Element of the Genes Encoding Vacuolar-Type ATPase Subunits and Assembly Factors.

Gene and genome comparison represent an invaluable tool to identify evolutionarily conserved sequences with possible functional significance. In this work, we have analyzed orthologous genes encoding subunits and assembly factors of the V-ATPase complex, an important enzymatic complex of the vacuolar and lysosomal compartments of the eukaryotic cell with storage and recycling functions, respectively, as well as the main pump in the plasma membrane that energizes the epithelial transport in insects. This study involves 70 insect species belonging to eight insect orders. We highlighted the conservation of a short sequence in the genes encoding subunits of the V-ATPase complex and their assembly factors analyzed with respect to their exon-intron organization of those genes. This study offers the possibility to study ultra-conserved regulatory elements under an evolutionary perspective, with the aim of expanding our knowledge on the regulation of complex gene networks at the basis of organellar biogenesis and cellular organization.

What Have We Learned in 30 Years of Investigations on Bari Transposons?

Transposable elements (TEs) have been historically depicted as detrimental genetic entities that selfishly aim at perpetuating themselves, invading genomes, and destroying genes. Scientists often co-opt "special" TEs to develop new and powerful genetic tools, that will hopefully aid in changing the future of the human being. However, many TEs are gentle, rarely unleash themselves to harm the genome, and bashfully contribute to generating diversity and novelty in the genomes they have colonized, yet they offer the opportunity to develop new molecular tools. In this review we summarize 30 years of research focused on the Bari transposons. Bari is a "normal" transposon family that has colonized the genomes of several Drosophila species and introduced genomic novelties in the melanogaster species. We discuss how these results have contributed to advance the field of TE research and what future studies can still add to the current knowledge.

Development of anti-somatostatin receptors CAR T cells for treatment of neuroendocrine tumors.

BACKGROUND: Neuroendocrine tumors (NETs) overexpress somatostatin receptors (SSTRs). METHODS: We developed a second-generation, ligand-based, anti-SSTR chimeric antigen receptor (CAR) incorporating the somatostatin analog octreotide in its extracellular moiety. RESULTS: Anti-SSTR CAR T cells exerted antitumor activity against SSTR+NET cell linesin vitro. The killing activity was highly specific, as demonstrated by the lack of CAR T cell reactivity against NET cells engineered to express mutated variants of SSTR2/5 by CRISPR/Cas9. When adoptively transferred in NSG mice, anti-SSTR CAR T cells induced significant antitumor activity against human NET xenografts. Although anti-SSTR CAR T cells could recognize the murine SSTRs as shown by their killing ability against murine NET cells, no obvious deleterious effects on SSTR-expressing organs such as the brain or the pancreas were observed in mice. CONCLUSIONS: Taken together, our results establish anti-SSTR CAR T cells as a potential candidate for early phase clinical investigations in patients with NETs. More broadly, the demonstration that a known peptide drug can direct CAR T cell targeting may streamline the potential utility of multiple peptide motifs and provide a blueprint for therapeutic applications in a variety of cancers.

Evidence of the Physical Interaction between Rpl22 and the Transposable Element Doc5, a Heterochromatic Transposon of Drosophila melanogaster.

Chromatin is a highly dynamic biological entity that allows for both the control of gene expression and the stabilization of chromosomal domains. Given the high degree of plasticity observed in model and non-model organisms, it is not surprising that new chromatin components are frequently described. In this work, we tested the hypothesis that the remnants of the Doc5 transposable element, which retains a heterochromatin insertion pattern in the melanogaster species complex, can be bound by chromatin proteins, and thus be involved in the organization of heterochromatic domains. Using the Yeast One Hybrid approach, we found Rpl22 as a potential interacting protein of Doc5. We further tested in vitro the observed interaction through Electrophoretic Mobility Shift Assay, uncovering that the N-terminal portion of the protein is sufficient to interact with Doc5. However, in situ localization of the native protein failed to detect Rpl22 association with chromatin. The results obtained are discussed in the light of the current knowledge on the extra-ribosomal role of ribosomal protein in eukaryotes, which suggests a possible role of Rpl22 in the determination of the heterochromatin in Drosophila.

Salting-Out Approach Is Worthy of Comparison with Ultracentrifugation for Extracellular Vesicle Isolation from Tumor and Healthy Models.

The role of extracellular vesicles (EVs) has been completely re-evaluated in the recent decades, and EVs are currently considered to be among the main players in intercellular communication. Beyond their functional aspects, there is strong interest in the development of faster and less expensive isolation protocols that are as reliable for post-isolation characterisations as already-established methods. Therefore, the identification of easy and accessible EV isolation techniques with a low price/performance ratio is of paramount importance. We isolated EVs from a wide spectrum of samples of biological and clinical interest by choosing two isolation techniques, based on their wide use and affordability: ultracentrifugation and salting-out. We collected EVs from human cancer and healthy cell culture media, yeast, bacteria and Drosophila culture media and human fluids (plasma, urine and saliva). The size distribution and concentration of EVs were measured by nanoparticle tracking analysis and dynamic light scattering, and protein depletion was measured by a colorimetric nanoplasmonic assay. Finally, the EVs were characterised by flow cytometry. Our results showed that the salting-out method had a good efficiency in EV separation and was more efficient in protein depletion than ultracentrifugation. Thus, salting-out may represent a good alternative to ultracentrifugation.

Field trial demonstrating phytoremediation of the military explosive RDX by XplA/XplB-expressing switchgrass.

The explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), a major component of munitions, is used extensively on military training ranges. As a result, widespread RDX pollution in groundwater and aquifers in the United States is now well documented. RDX is toxic, but its removal from training ranges is logistically challenging, lacking cost-effective and sustainable solutions. Previously, we have shown that thale cress (Arabidopsis thaliana) engineered to express two genes, xplA and xplB, encoding RDX-degrading enzymes from the soil bacterium Rhodococcus rhodochrous 11Y can break down this xenobiotic in laboratory studies. Here, we report the results of a 3-year field trial of XplA/XplB-expressing switchgrass (Panicum virgatum) conducted on three locations in a military site. Our data suggest that XplA/XplB switchgrass has in situ efficacy, with potential utility for detoxifying RDX on live-fire training ranges, munitions dumps and minefields.