Protocol for aerial trapping and analyses of candidate pheromone compounds released by moths via gas chromatography-mass spectrometry.

Detection of pheromones is pivotal to chemical ecology and agronomy; however, analytic detection of the volatile pheromone components from odorized air is highly challenging. Here, we introduce a protocol for the detection of airborne pheromones from female moths, which are key models for chemosensory studies. We describe a step-by-step guide from pheromone collection to quantitative estimation of pheromone components. We also detail procedures for gas chromatography-mass spectrometry (GC-MS) analysis. This protocol has potential applications beyond chemosensory research, particularly in environmental chemistry. For complete details on the use and execution of this protocol, please refer to Ghosh et al.1.

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.

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.

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.

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.

The Interaction between Reactive Peritoneal Mesothelial Cells and Tumor Cells via Extracellular Vesicles Facilitates Colorectal Cancer Dissemination.

Advanced colorectal cancer (CRC) is highly metastatic and often results in peritoneal dissemination. The extracellular vesicles (EVs) released by cancer cells in the microenvironment are important mediators of tumor metastasis. We investigated the contribution of EV-mediated interaction between peritoneal mesothelial cells (MCs) and CRC cells in generating a pro-metastatic environment in the peritoneal cavity. Peritoneal MCs isolated from peritoneal lavage fluids displayed high CD44 expression, substantial mesothelial-to-mesenchymal transition (MMT) and released EVs that both directed tumor invasion and caused reprogramming of secretory profiles by increasing TGF-ß1 and uPA/uPAR expression and MMP-2/9 activation in tumor cells. Notably, the EVs released by tumor cells induced apoptosis by activating caspase-3, peritoneal MC senescence, and MMT, thereby augmenting the tumor-promoting potential of these cells in the peritoneal cavity. By using pantoprazole, we reduced the biogenesis of EVs and their pro-tumor functions. In conclusion, our findings provided evidence of underlying mechanisms of CRC dissemination driven by the interaction of peritoneal MCs and tumor cells via the EVs released in the peritoneal cavity, which may have important implications for the clinical management of patients.

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.

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.

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.

Functional characterization of the Bari1 transposition system.

The transposons of the Bari family are mobile genetic elements widespread in the Drosophila genus. However, despite a broad diffusion, virtually no information is available on the mechanisms underlying their mobility. In this paper we report the functional characterization of the Bari elements transposition system. Using the Bari1 element as a model, we investigated the subcellular localization of the transposase, its physical interaction with the transposon, and its catalytic activity. The Bari1 transposase localized in the nucleus and interacted with the terminal sequences of the transposon both in vitro and in vivo, however, no transposition activity was detected in transposition assays. Profiling of mRNAs expressed by the transposase gene revealed the expression of abnormal, internally processed transposase transcripts encoding truncated, catalytically inactive transposase polypeptides. We hypothesize that a post-transcriptional control mechanism produces transposase-derived polypeptides that effectively repress transposition. Our findings suggest further clues towards understanding the mechanisms that control transposition of an important class of mobile elements, which are both an endogenous source of genomic variability and widely used as transformation vectors/biotechnological tools.

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.

Condylar position indicator and T-scan system II in clinical evaluation of temporomandibular intracapsular disease.

INTRODUCTION: The pathogenesis of temporomandibular joint intracapsular disease (TMJI) is multifactorial and its diagnosis is not easy. In this work authors show two types of clinical analysis: the Condylar Position Indicator (CPI) and T-Scan 2 system. MATERIAL AND METHODS: Twenty patients (mean age of 24.5 years) with TMJI problem and 10 healthy matched subjects (mean age: 25.4 years) were selected. Analysis of TMJI was performed on each patient by means of Condylar Position Indicator (CPI) and T-Scan System II tests. RESULTS: Eight patients presented vertical symmetrical condylar distraction greater than healthy subjects (P-value<0.001). T-Scan showed a difference of Percentage of Force (POF) not greater than 5%. Seven patients showed sagittal shift greater than healthy subject (P-value<0.001). T-Scan records showed a difference of POF greater than 5%. Five non-healthy subjects presented sagittal, vertical, transverse shift greater than healthy subjects (P-value<0.001). T-Scan records show a difference of POF greater than 5%. CONCLUSION: In this work authors present a new method of analysis. CPI indicates discrepancy of the condyle position in CO from CR and T-Scan allows the operator to study all teeth contacts and occlusal forces taking place during dynamic jaw movement.

Molecular genetic linkage maps for allotetraploid Leymus wildryes (Gramineae: Triticeae).

Molecular genetic maps were constructed for two full-sib populations, TTC1 and TTC2, derived from two Leymus triticoides x Leymus cinereus hybrids and one common Leymus triticoides tester. Informative DNA markers were detected using 21 EcoRI-MseI and 17 PstI-MseI AFLP primer combinations, 36 anchored SSR or STS primer pairs, and 9 anchored RFLP probes. The 164-sib TTC1 map includes 1069 AFLP markers and 38 anchor loci in 14 linkage groups spanning 2001 cM. The 170-sib TTC2 map contains 1002 AFLP markers and 36 anchor loci in 14 linkage groups spanning 2066 cM. Some 488 homologous AFLP loci and 24 anchor markers detected in both populations showed similar map order. Thus, 1583 AFLP markers and 50 anchor loci were mapped into 14 linkage groups, which evidently correspond to the 14 chromosomes of allotetraploid Leymus (2n = 4x = 28). Synteny of two or more anchor markers from each of the seven homoeologous wheat and barley chromosomes was detected for 12 of the 14 Leymus linkage groups. Moreover, two distinct sets of genome-specific STS markers were identified in these allotetraploid Leymus species. These Leymus genetic maps and populations will provide a useful system to evaluate the inheritance of functionally important traits of two divergent perennial grass species.