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Objectives: To examine the association between muscle strength and cardiorespiratory fitness (CRF) with all-cause and cancer-specific mortality in patients diagnosed with cancer, and whether these associations are affected by type and/or stage of cancer., Method: A systematic review with meta-analysis was carried out. Five bibliographic databases were searched to August 2023., Results: Forty-two studies were included (n=46 694). Overall, cancer patients with high muscle strength or CRF levels (when dichotomised as high vs low) had a significant reduction in risk of all-cause mortality by 31-46% compared with those with low physical fitness levels. Similarly, a significant 11% reduction was found for change per unit increments in muscle strength. In addition, muscle strength and CRF were associated with an 8-46% reduced risk of all-cause mortality in patients with advanced cancer stages, and a 19-41% reduced risk of all-cause mortality was observed in lung and digestive cancers. Lastly, unit increments in CRF were associated with a significant 18% reduced risk of cancer-specific mortality., Conclusion: High muscle strength and CRF were significantly associated with a lower risk of all-cause mortality. In addition, increases in CRF were associated with a reduced risk of cancer-specific mortality. These fitness components were especially predictive in patients with advanced cancer stages as well as in lung and digestive cancers. This highlights the importance of assessing fitness measures for predicting mortality in cancer patients. Given these findings, tailored exercise prescriptions to improve muscle strength and CRF in patients with cancer may contribute to reducing cancer-related mortality., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2025. No commercial re-use. See rights and permissions. Published by BMJ Group.)

Addressing the global challenge of ensuring access to safe drinking water, especially in developing countries, demands cost-effective, eco-friendly, and readily available technologies. The persistence, toxicity, and bioaccumulation potential of organic pollutants arising from various human activities pose substantial hurdles. While high-performance liquid chromatography coupled with high-resolution mass spectrometry (HPLC-HRMS) is a widely utilized technique for identifying pollutants in water, the multitude of structures for a single elemental composition complicates structural identification. While current HRMS and MS/MS databases often can provide hits for known molecules, these are often erroneous or misleading when authentic standards are unavailable. In this research, a machine-learning algorithm is developed to support the structural elucidation of small organic pollutants in water, with a focus on (carbon, oxygen, and hydrogen-based) molecules weighing less than 500 Da. The approach relies on a comparison of the experimental and predicted retention of the possible structures of unknowns for which an elemental composition was obtained by HRMS. A promising novelty is thereby the improved removal of erroneous structures via the combination of the retention information obtained from two reversed-phase-based stationary phases, depicting different selectivities (octadecylsilica, C18 and pentafluorphenylsilica, F5). The study translates retention times into retention indices for instrument independence and transferability across diverse HPLC-HRMS systems. The predictive algorithm, utilizing retention data and molecular descriptors, accurately predicts retention indices and proves its utility by eliminating incorrect structural formulas through a 2-stationary phase intersection-based filtration. Using a data set of 100 training compounds and 16 external test set compounds, two Multiple Linear Regression (MLR), MLR-C18 and MLR-F5 models were developed, employing the 16 most influential descriptors, out of 5666 screened. MLR-C18 achieves precise RI predictions, R 2 = 0.97, RMSE = 36, MAE = 26, while MLR-F5, though slightly less accurate, maintains a performance with R 2 = 0.96, RMSE = 44, MAE = 34. The intersection-based filtration (within ±1.5σ) showed the elimination of more than 70% of impossible structures for a given elemental composition. The model was further implemented in the identification of a drinking water sample to prove its potential. This tool holds significant promise for supporting water quality management and sustainable practices, contributing to faster structural identification of unknown organic micropollutants in water.

Accurate monitoring of pesticide residues at minimal concentrations is imperative for adherence to stringent regulatory standards in numerous countries. This study presents an innovative methodology employing comprehensive two-dimensional liquid chromatography coupled with high-resolution mass spectrometry (LC × LC-HRMS). The approach ensures high sensitivity and selectivity in detecting targeted compounds. A pivotal component of this methodology is the utilization of per-aqueous liquid chromatography (PALC) as the first dimension, facilitating the use of water-based mobile phases and addressing solvent mismatch issues. The second dimension employs reversed-phase liquid chromatography (RPLC), enhancing the separation of compounds. PALC proves instrumental in refocusing and enables the practical application of narrow-diameter columns (1.5 mm I.D.). This column design permits a direct split-free connection of the LC × LC to an electrospray-based mass spectrometer (ESI-MS), contributing to heightened sensitivity. The MS acquisition is performed in a targeted single-ion monitoring mode, ensuring reliable quantification and identification of the pesticide compounds. A comprehensive evaluation of key performance metrics, including signal-to-noise ratio, limit of detection, and response linearity, is conducted. The methodology achieves a limit of detection below the ng mL -1 and exhibits response linearity within the concentration range of 1-100 ng mL -1 . The robustness of the approach is further demonstrated through intra-day and inter-day repeatability validations. Furthermore, the platform is finally tested on a surface water sample. This study not only introduces an advanced analytical methodology for pesticide multi-residue analysis but also underscores the significance of PALC in enhancing sensitivity by facilitating the use of smaller-diameter columns and water-based mobile phases, along with the role of RPLC in enhancing separation. The proposed approach showcases promising results in achieving detection limits that match the stringent regulatory standards and reliable quantification for effective pesticide residue monitoring., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Temperature-responsive liquid chromatography (TRLC) offers a promising alternative to reversed-phase liquid chromatography (RPLC) for environmentally friendly analytical techniques by utilizing pure water as a mobile phase, eliminating the need for harmful organic solvents. TRLC columns, packed with temperature-responsive polymers coupled to silica particles, exhibit a unique retention mechanism influenced by temperature-induced polymer hydration. An investigation of the physicochemical parameters driving separation at high and low temperatures is crucial for better column manufacturing and selectivity control. Assessment of predictability using a dataset of 139 molecules analyzed at different temperatures elucidated the molecular descriptors (MDs) relevant to retention mechanisms. Linear regression, support vector regression (SVR), and tree-based ensemble models were evaluated, with no standout performer. The precision, accuracy, and robustness of models were validated through metrics, such as r and mean absolute error (MAE), and statistical analysis. At 45 ∘ C , logP predominantly influenced retention, akin to reversed-phase columns, while at 5 ∘ C , complex interactions with lipophilic and negative MDs, along with specific functional groups, dictated retention. These findings provide deeper insights into TRLC mechanisms, facilitating method development and maximizing column potential., (© 2024. The Author(s).)

This study explores the possibilities offered by temperature-responsive liquid chromatography (TRLC) based comprehensive 2-dimensional liquid chromatography in combination with reversed-phase liquid chromatography (RPLC) for the analysis of degradation products formed upon oxidative treatment of persistent organic pollutants, in this case exemplified through carbamazepine (CBZ). The TRLC×RPLC combination offers the possibility to overcome peak overlap and incomplete separation encountered in 1D approaches, while the transfer of the purely aqueous mobile phase leads to refocusing of all analytes on the second dimension column. Consequently, this allows for about method-development free and hence, easier LC×LC. The study focuses on the oxidative degradation of CBZ, a compound of environmental concern due to its persistence in water bodies. The TRLC×RPLC combination effectively separates and identifies CBZ and its degradation products, while offering improved selectivity over the individual TRLC or RPLC separations. This allows gathering more understanding of the degradation cascade and allows real-time monitoring of the appearance and disappearance of various degradation products. The compatibility with high-resolution mass spectrometry is last shown, enabling identification of 21 CBZ-related products, nine of which were not previously reported in CBZ degradation studies. The approach's simplicity, optimization-free aspects, and ease of use make it a promising tool for the analysis of degradation pathways in environmental contaminants., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Frederic Lynen reports financial support was provided by EU Framework Program for Research and Innovation Horizon 2020. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Slightly different reaction conditions afforded two distinct cavity-shaped cis-chelating diphosphanes from the same starting materials, namely diphenyl(2-phosphanylphenyl)phosphane and an α-cyclodextrin-derived dimesylate. Thanks to their metal-confining properties, the two diphosphanes form only mononuclear [CuX(PP)] complexes (X=Cl, Br, or I) with the tricoordinated metal ion located just above the center of the cavity. The two series of Cu I complexes display markedly different luminescence properties that are both influenced by the electronic properties of the ligand and the unique steric environment provided by the cyclodextrin (CD) cavity. The excited state lifetimes of all complexes are significantly longer than those of the cavity-free analogues suggesting peculiar electronic effects that affect radiative deactivation constants. The overall picture stemming from absorption and emission data suggests close-lying charge-transfer (MLCT, XLCT) and triplet ligand-centered (LC) excited states., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Although once regarded as a unique human feature, tool-use is widespread in the animal kingdom. Some of the most proficient tool-users are our closest living relatives, chimpanzees. These repertoires however consist primarily of tool use, rather than tool manufacture (for later use). Furthermore, most populations of chimpanzees use organic materials, such as sticks and leaves, rather than stones as tools. This distinction may be partly ecological, but it is also important as chimpanzees are often used as models for the evolution of human material culture, the oldest traces of which consist of manufactured sharp stone tools (so-called "flakes"). Thus, examining the conditions (if any) under which chimpanzees may develop flake manufacture and use can provide insight into the drivers of these behaviours in our own lineage. Previous studies on non-human apes' ability to make and use flakes focused on enculturated apes, giving them full demonstrations of the behaviour immediately, without providing social information on the task in a stepwise manner. Here we tested naïve, captive chimpanzees (N = 4; three potentially enculturated and one unenculturated subject) in a social learning experimental paradigm to investigate whether enculturated and/or unenculturated chimpanzees would develop flake making and use after social information of various degrees (including a human demonstration) was provided in a scaffolded manner. Even though social learning opportunities were provided, neither the unenculturated subject nor any of the potentially enculturated subjects made or used flakes, in stark contrast to previous studies with enculturated apes. These data suggest that flake manufacture and use is outside of our tested group of captive chimpanzees' individual and social learning repertoires. It also suggests that high levels of enculturation alongside human demonstrations (and/or training) may be required before captive chimpanzees can develop this behaviour., (© 2023. The Author(s).)

Temperature-responsive liquid chromatography (TRLC) offers an alternative for retention and selectivity optimisation in HPLC. This approach thereby exploits temperature gradients on stimuli-responsive stationary phases and forfeits the necessity for solvent gradients, allowing analyses to be performed using aqueous mobile phases. Consequently, it can be employed as a green alternative to reversed-phase separations. However, current production to obtain temperature-responsive columns inherently require dedicated column packing processes with polymer-modified particles. To facilitate the development of temperature-responsive phases, a flow-through modification procedure was developed allowing on-column modification of aminopropyl silica columns. Three columns were manufactured using this novel flow-through approach, which achieved identical column efficiencies compared to existing TRLC column. Demonstrating the possibility of bypassing the dedicated packing processes without losing efficiency. Additionally, it was observed that flow-through produced columns yielded higher retention at elevated temperatures despite their reduced carbon load. Further investigation of the carbon load revealed the presence of stationary phase gradients, whose influence was studied via novel developed retention experiments, which revealed a negligible change reduction in retention upon a change of polymer modification from 19.8% to 14.5%. However, further decrease from 14.5% to 12.3% resulted in a larger change. Interestingly, a further enhancement in apparent plate numbers was observed when operating the column under a reversed flow, yielding an increasing stationary phase gradient. This on-column modification procedure demonstrates the potential for modification of existing (commercial) packed columns to achieve temperature-responsive phases without loss of efficiency or retention. Thus, not only facilitating accessibility to temperature-responsive phases, but also aiding with development of further generations of temperature-responsive phases by removing the need for packing optimisation. Additionally, a novel experiment was set up to easily investigate the effect of inhomogeneous stationary phases retention., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowedgements The authors want to acknowledge the FWO and FNRS for funding part of this research through an Excellence of Science grant (30897864) and FWO grant (G0D0218N), as well as co-funding from a BOF-UGent grant (01J15417)., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Subjects with pathogenic (PV) and likely pathogenic (LPV) FLCN variants have an increased risk of manifesting benign and malignant disorders that are related to Birt-Hogg-Dubé syndrome (BHDS): an autosomal dominantly inherited disorder whose severity can vary significantly. Renal cell carcinoma (RCC) development in BHD (Birt-Hogg-Dubé) patients has a very high incidence; thus, identifying this rare syndrome at early stages and preventing metastatic spread is crucial. Over the last decade, the advancement of Next Generation Sequencing (NGS) and the implementation of multigene panels for hereditary cancer syndromes (HCS) have led to a subsequent focus on additional genes and variants, including those of uncertain significance (VUS). Here, we describe a novel FLCN variant observed in a subject manifesting disorders that were suspected to be related to BHDS and with a family history of multiple cancers.

This paper reports the synthesis, structure, photophysical, and optoelectronic properties of five eight-coordinate Europium(III) ternary complexes, namely, [Eu(hth) 3 (L) 2 ], bearing 4,4,5,5,6,6,6-heptafluoro-1-(2-thienyl)-1,3-hexanedione (hth) as a sensitizer and L = H 2 O ( 1 ), dpso (diphenyl sulphoxide, 2 ), dpsoCH 3 (4,4'-dimethyl diphenyl sulfoxide, 3 ), dpsoCl (bis(4-chlorophenyl)sulphoxide, 4 ), and tppo (triphenylphosphine oxide, 5 ) as co-ligands. The NMR and the crystal structure analysis confirmed the eight-coordinate structures of the complexes in solution and in a solid state. Upon UV-excitation on the absorption band of the β-diketonate ligand hth, all complexes showed the characteristic bright red luminescence of the Europium ion. The tppo derivative ( 5 ) displayed the highest quantum yield (up to 66%). As a result, an organic light-emitting device, OLED, was fabricated with a multi-layered structure-ITO/MoO 3 /mCP/SF3PO:[complex 5 ] (10%)/TPBi:[complex 5 ] (10%)/TmPyPB/LiF/Al-using complex 5 as the emitting component.

PVs and LPVs in BRCA1/2 genes are correlated to a high risk of developing breast cancer and/or ovarian cancer (Hereditary Breast and Ovarian Cancer syndrome, HBOC); additionally, in recent years, an increasing number of BRCA 1/2 variants have been identified and associated with pancreatic cancer. Epidemiologic studies have highlighted that inherited factors are involved in 10% to 20% of PCs, mainly through deleterious variants of BRCA2 . The frequency of BRCA1/2 germline alterations fluctuates quite a lot among different ethnic groups, and the estimated rate of PVs/LPVs variants in Italian HBOC families is not very accurate, according to different reports. The aim of our study is to describe the prevalence of a BRCA2 PV observed in a selected cohort of HBOC patients and their relatives, whose common origin is the eastern coast of Emilia Romagna, a region of Italy. This study provides insight into the frequency of the variant detected in this area and provides evidence of an increased risk of pancreatic and breast cancer, useful for genetic counseling and surveillance programs., Competing Interests: The authors declare no conflict of interest.

Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

A β-cyclodextrin-based diphosphane with metal-confining properties was efficiently synthesized thanks to an unprecedented Smiles-like rearrangement of diphenyl-(2-phosphanylphenyl)phosphane in the presence of excess n-BuLi. The cis-chelating bidentate ligand is capable of forming very stable heteroleptic [Cu(NN)(PP)] + complexes in which a metal-bound diimine ligand (bpy, phen, or mmp) is located within the cyclodextrin cavity. As a result of ligand encapsulation, flattening of the metal tetrahedral geometry in the excited state is disfavored, thereby resulting in enhanced luminescent properties., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Refractive index detection (RID) is attractive because it allows approaching the benefits of universal detection with liquid chromatography, by which ideally standard independent calibration and hence compound independent quantification becomes possible. Nevertheless, the implementation of RID has remained limited as it offers poor detection sensitivity while only being compatible with isocratic mobile phases. The implementation of compositional solvent gradients has remained prohibitively challenging in commercial HPLC-RID systems due to the resulting drastic alterations in refractive index and extreme baseline drift. While the refractive index is also highly dependent on temperature, more leeway appears possible to mitigate the problem, particularly when the used temperature gradients can be limited. Temperature-responsive liquid chromatography (TRLC) allows obtaining isocratic reversed phase type of separations, whereby retention is modulated via temperature changes ∼ 15 °C-20 °C above and below the polymer conversion temperature. Elution profiles, reminiscent of what can be obtained with solvent gradients in conventional RPLC, can then be obtained by enacting downwards temperature gradients on the columns. This work comprises a proof-of-principle to illustrate the possibilities of combining thermal gradient TRLC with RID. The observed baseline drift appeared thereby very minor (<5 nRIU min -1 ), and hence easily controllable. Short chain fatty acids are used as representative compounds to assess this new approach. Overlapping calibration lines are accordingly obtained for all fatty acids between butyric and decanoic acid., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Heteroleptic cyclometalated iridium (III) complexes ( 1 - 3 ) containing di-pyridylamine motifs were prepared in a stepwise fashion. The presence of the di-pyridylamine ligands tunes their electronic and optical properties, generating blue phosphorescent emitters at room temperature. Herein we describe the synthesis of the mononuclear iridium complexes [Ir(ppy) 2 (DPA)][OTf] ( 1 ), (ppy = phenylpyridine; DPA = Dipyridylamine) and [Ir(ppy) 2 (DPA-PhI)][OTf] ( 2 ), (DPA-PhI = Dipyridylamino-phenyliodide). Moreover, the dinuclear iridium complex [Ir(ppy) 2 ( L )Ir(ppy) 2 ][OTf] 2 ( 3 ) containing a rigid angular ligand " L = 3,5-bis[4-(2,2'-dipyridylamino)phenylacetylenyl]toluene" and displaying two di-pyridylamino groups was also prepared. For comparison purposes, the related dinuclear rhodium complex [Rh (ppy) 2 ( L )Rh(ppy) 2 ][OTf] 2 ( 4 ) was also synthesized. The x-ray molecular structure of complex 2 was reported and confirmed the formation of the target molecule. The rhodium complex 4 was found to be emissive only at low temperature; in contrast, all iridium complexes 1 - 3 were found to be phosphorescent in solution at 77 K and room temperature, displaying blue emissions in the range of 478-481 nm.

Introduction: Retinoblastoma (Rb) is the most common ocular paediatric malignancy and is caused by a mutation of the two alleles of the tumor suppressor gene, RB1 . The tumor microenvironment (TME) represents a complex system whose function is not yet well defined and where microvesicles, such as exosomes, play a key role in intercellular communication. Micro-RNAs (mRNAs) have emerged as important modifiers of biological mechanisms involved in cancer and been able to regulate tumor progression., Methods: Co-culture of monocytes with retinoblastoma cell lines, showed a significant growth decrease. Given the interaction between Rb cells and monocytes, we investigated the role of the supernatant in the cross-talk between cell lines, by taking the product of the co-culture and then using it as a culture medium for Rb cells., Results: miR-142-3p showed to be particularly over-expressed both in the Rb cell line and in the medium used for their culture, comparing to control cell line and the normal supernatant, respectively. Therefore, we provided evidence that miR-142-3p is released by monocytes in the co-culture medium's exosomes and that it is subsequently up-taken by Rb cells, causing the inhibition of proliferation of Rb cell line by affecting cell cycle progression., Conclusion: This study highlights the role of exosomic miR-142-3p in the TME of Rb and identifies new molecular targets, which are able to control tumor growth aiming the development of a forward-looking miR-based strategy., Competing Interests: The authors report no conflicts of interest in this work., (© 2022 Plousiou et al.)

A novel Cu(I) cluster compound has been synthesized by reacting CuI with the 2,2'-dithiobis(5-nitropyridine) ligand under solvothermal conditions. During the reaction, the original ligand breaks into the 5-nitropyridine-2-thiolate moiety, which acts as the coordinating ligand with both N- and S-sites, leading to a distorted octahedral Cu 6 S 6 cluster. The structure has been determined by single-crystal X-ray diffraction and FT-IR analysis, and the photophysical properties have been determined in the solid state by means of steady-state and time-resolved optical techniques. The cluster presents a near-infrared emission showing an unusual temperature dependence: when passing from 77 to 298 K, a blue-shift of the emission band is observed, associated with a decrease in its intensity. Time-dependent-density functional theory calculations suggest that the observed behavior can be ascribed to a complex interplay of excited states, basically in the triplet manifold., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Birt-Hogg-Dubé syndrome (BHDS) is a rare autosomal dominant inherited disorder caused by a mutation in folliculin ( FLCN ) gene transmitted via germline autosomal dominant pattern. Patients with this syndrome have an increased susceptibility to renal cell carcinoma, lung cysts, spontaneous pneumothorax, and benign skin hamartomas, and its diagnosis is not easy and consequently underestimated. Several mutations have been identified in FLCN gene, among which the majority of alterations are frameshift (insertion/deletion), nonsense, or splice-site mutations that generally produce unfunctional truncated FLCN proteins. Our aim is to present a case of a BHDS family whose proband is a 56-year-old patient who has been experiencing multiple disorders, has an FLCN genetic mutation, and has also been identified to have a pathogenic variant in BRCA2 gene. Our further purpose is to emphasize the importance of the next-generation sequencing (NGS) approach to identify potential multiple germline mutations in complex and rare oncologic disorders, allowing strict and more targeted cancer screening programs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bandini, Cangini, Arcangeli, Ravegnani, Andreotti, Prisinzano, Pastorino, Martinelli, Falcini, Calistri, Zampiga and Danesi.)

Phenolic compounds are an interesting class of natural products because of their proposed contribution to health benefits of foods and beverages and as a bio-source of organic (aromatic) building blocks. Phenolic extracts from natural products are often highly complex and contain compounds covering a broad range in molecular properties. While many 1D-LC and mass spectrometric approaches have been proposed for the analysis of phenolics, this complexity inevitably leads to challenging identification and purification. New insights into the composition of phenolic extracts can be obtained through online comprehensive two-dimensional liquid chromatography (LC × LC) coupled to photodiode array and mass spectrometric detection. However, several practical hurdles must be overcome to achieve high peak capacities and to obtain robust methods with this technique. In many LC × LC configurations, refocusing of analytes at the head of the 2 D column is hindered by the high eluotropic strength of the solvent transferred from the 1 D to the 2 D, leading to peak breakthrough or broadening. LC × LC combinations whereby a purely aqueous mobile phase is used in the 1 D and RPLC is used in the 2 D are unaffected by these phenomena, leading to more robust methods. In this contribution, the combination of temperature-responsive liquid chromatography (TRLC) with RPLC is used for the first time for the analysis of phenolic extracts of natural origin to illustrate the potential of this alternative combination for natural product analyses. The possibilities of the combination are investigated through analysis of wine extracts by TRLC × RPLC-DAD and TRLC × RPLC-ESI-MS., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Here, we monitored the evolution of CTCs spread in 11 patients affected by locally advanced EC who were undergoing therapy., Methods: In this perspective study, we designed multiple blood biopsies from individual patients: before and after neoadjuvant chemo-radio therapy and after surgery. We developed a multi-target array, named Grab-all assay, to estimate CTCs for their epithelial (EpCAM/E-Cadherin/Cytokeratins) and mesenchymal/stem (N-Cadherin/CD44v6/ABCG2) phenotypes. Identified CTCs were isolated as single cells by DEPArray, subjected to whole genome amplification, and copy number aberration (CNA) profiles were determined. Through bioinformatic analysis, we assessed the genomic imbalance of single CTCs, investigated specific focal copy number changes previously reported in EC and aberrant pathways using enrichment analysis., Results: Longitudinal monitoring allowed the identification of CTCs in at least one time-point per patient. Through single cell CNA analysis, we revealed that CTCs showed significantly dynamic genomic imbalance during treatment. Individual CTCs from relapsed patients displayed a higher degree of genomic imbalance relative to disease-free patients' groups. Genomic aberrations previously reported in EC occurred mostly in post-neoadjuvant therapy CTCs. In-depth analysis showed that networks enrichment in all time-point CTCs were inherent to innate immune system. Transcription/gene regulation, post-transcriptional and epigenetic modifications were uniquely affected in CTCs of relapsed patients., Conclusions: Our data add clues to the comprehension of the role of CTCs in EC aggressiveness: chromosomal aberrations on genes related to innate immune system behave as relevant to the onset of CTC-status, whilst pathways of transcription/gene regulation, post-transcriptional and epigenetic modifications seem linked to patients' outcome.

Background: Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. Chemotherapy, the treatment of choice in non-operable cases, achieves a dismal success rate, raising the need for new therapeutic options. In about 25% of NSCLC, the activating mutations of the KRAS oncogene define a subclass that cannot benefit from tyrosine kinase inhibitors (TKIs). The tumor suppressor miR-16 is downregulated in many human cancers, including NSCLC. The main objectives of this study were to evaluate miR-16 treatment to restore the TKI sensitivity and compare its efficacy to MEK inhibitors in KRAS -mutated NSCLC., Methods: We performed in vitro and in vivo studies to investigate whether miR-16 could be exploited to overcome TKI resistance in KRAS-mutated NSCLC. We had three goals: first, to identify the KRAS downstream effectors targeted by mir-16, second, to study the effects of miR-16 restoration on TKI resistance in KRAS-mutated NSCLC both in vitro and in vivo, and finally, to compare miR-16 and the MEK inhibitor selumetinib in reducing KRAS-mutated NSCLC growth in vitro and in vivo., Results: We demonstrated that miR-16 directly targets the three KRAS downstream effectors MAPK3 , MAP2K1 , and CRAF in NSCLC, restoring the sensitivity to erlotinib in KRAS -mutated NSCLC both in vitro and in vivo. We also provided evidence that the miR-16-erlotinib regimen is more effective than the selumetinib-erlotinib combination in KRAS -mutated NSCLC., Conclusions: Our findings support the biological preclinical rationale for using miR-16 in combination with erlotinib in the treatment of NSCLC with KRAS -activating mutations.

Polycythemia Vera (PV) is a myeloproliferative neoplasm with increased risk of thrombosis and progression to myelofibrosis. Chronic inflammation is commonly observed in myeloproliferative neoplasms including PV. The inflammatory network includes the extracellular vesicles (EVs), which play a role in cell-cell communication. Recent evidence points to circulating microbial components/microbes as potential players in hemopoiesis regulation. To address the role of EVs in PV, here we investigated phenotype and microbial DNA cargo of circulating EVs through multidimensional analysis. Peripheral blood and feces were collected from PV patients (n=38) and healthy donors (n=30). Circulating megakaryocyte (MK)- and platelet (PLT)-derived EVs were analyzed by flow cytometry. After microbial DNA extraction from feces and isolated EVs, the 16S rDNA V3-V4 region was sequenced. We found that the proportion of circulating MK-derived EVs was significantly decreased in PV patients as compared with the healthy donors. By contrast, the proportion of the PLT-derived EVs was increased. Interestingly, PV was also associated with a microbial DNA signature of the isolated EVs with higher diversity and distinct microbial composition than the healthy counterparts. Of note, increased proportion of isolated lipopolysaccharide-associated EVs has been demonstrated in PV patients. Conversely, the gut microbiome profile failed to identify a distinct layout between PV patients and healthy donors. In conclusion, PV is associated with circulating EVs harbouring abnormal phenotype and dysbiosis signature with a potential role in the (inflammatory) pathogenesis of the disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Barone, Barone, Ricci, Auteri, Corradi, Fabbri, Papa, Bandini, Cenacchi, Tazzari, Vianelli, Turroni, Cavo, Palandri, Candela and Catani.)

Breast cancer (BC) is the most commonly diagnosed malignant tumor in women worldwide, and the leading cause of cancer death in the female population. The percentage of patients experiencing poor prognosis along with the risk of developing metastasis remains high, also affecting the resistance to current main therapies. Cancer progression and metastatic development are no longer due entirely to their intrinsic characteristics, but also regulated by signals derived from cells of the tumor microenvironment. Extracellular vesicles (EVs) packed with DNA, RNA, and proteins, are the most attractive targets for both diagnostic and therapeutic applications, and represent a decisive challenge as liquid biopsy-based markers. Here we performed a study based on a multiplexed phenotyping flow cytometric approach to characterize BC-derived EVs from BC patients and cell lines, through the detection of multiple antigens. Our data reveal the expression of EVs-related biomarkers derived from BC patient plasma and cell line supernatants, suggesting that EVs could be exploited for characterizing and monitoring disease progression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bandini, Rossi, Scarpi, Gallerani, Vannini, Salvi, Azzali, Melloni, Salucci, Battistelli, Serra, Maltoni, Cho and Fabbri.)

Polycythemia vera is a myeloproliferative neoplasm with increased risk of thrombosis and progression to myelofibrosis. However, no disease-specific risk factors have been identified so far. Circulating extracellular vesicles (EVs) are mostly of megakaryocyte (MK-EVs) and platelet (PLT-EVs) origin and, along with phosphatidylethanolamine (PE)-EVs, play a role in cancer and thrombosis. Interestingly, circulating microbial components/microbes have been recently indicated as potential modifiers of inflammation and coagulation. Here, we investigated phenotype and microbial DNA cargo of EVs after isolation from the plasma of 38 patients with polycythemia vera. Increased proportion of MK-EVs and reduced proportion of PLT-EVs identify patients with thrombosis history. Interestingly, EVs from patients with thrombosis history were depleted in Staphylococcus DNA but enriched in DNA from Actinobacteria members as well as Anaerococcus . In addition, patients with thrombosis history had also lower levels of lipopolysaccharide-associated EVs. In regard to fibrosis, along with increased proportion of PE-EVs, the EVs of patients with marrow fibrosis were enriched in DNA from Collinsella and Flavobacterium . Here, we identified a polycythemia-vera-specific host/microbial EV-based signature associated to thrombosis history and marrow fibrosis. These data may contribute to refining PV prognosis and to identifying novel druggable targets.

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Nut-cracking with hammer tools (henceforth: nut-cracking) has been argued to be one of the most complex tool-use behaviors observed in nonhuman animals. So far, only chimpanzees, capuchins, and macaques have been observed using tools to crack nuts in the wild (Boesch and Boesch, 1990; Gumert et al., 2009; Mannu and Ottoni, 2009). However, the learning mechanisms behind this behavior, and the extent of nut-cracking in other primate species are still unknown. The aim of this study was two-fold. First, we investigated whether another great ape species would develop nut-cracking when provided with all the tools and appropriate conditions to do so. Second, we examined the mechanisms behind the emergence of nut-cracking by testing a naïve sample. Orangutans (Pongo abelii and Pongo pygmaeus) have the second most extensive tool-use repertoire among the great apes (after chimpanzees) and show flexible problem-solving capacities. Orangutans have not been observed cracking nuts in the wild, however, perhaps because their arboreal habits provide limited opportunities for nut-cracking. Therefore, orangutans are a valid candidate species for the investigation of the development of this behavior. Four nut-cracking-naïve orangutans at Leipzig zoo (P. abelii; M age  = 16; age range = 10-19; 4F; at the time of testing) were provided with nuts and hammers but were not demonstrated the nut-cracking behavioral form. Additionally, we report data from a previously unpublished study by one of the authors (Martina Funk) with eight orangutans housed at Zürich zoo (six P. abelii and two P. pygmaeus; M age  = 14; age range = 2-30; 5F; at the time of testing) that followed a similar testing paradigm. Out of the twelve orangutans tested, at least four individuals, one from Leipzig (P. abelii) and three from Zürich (P. abelii and P. pygmaeus), spontaneously expressed nut-cracking using wooden hammers. These results demonstrate that nut-cracking can emerge in orangutans through individual learning and certain types of non-copying social learning., (© 2021 The Authors. American Journal of Primatology published by Wiley Periodicals LLC.)

Background : Despite substantial research on early hominin lithic technologies, the learning mechanisms underlying flake manufacture and use are contested. To draw phylogenetic inferences on the potential cognitive processes underlying the acquisition of both of these abilities in early hominins, we investigated if and how one of our closest living relatives, chimpanzees ( Pan troglodytes ), could learn to make and use flakes. Methods : Across several experimental conditions, we tested eleven task-naïve chimpanzees (unenculturated n=8, unknown status n=3) from two independent populations for their abilities to spontaneously make and subsequently use flakes as well as to use flakes made by a human experimenter. Results : Despite the fact that the chimpanzees seemed to understand the requirements of the task, were sufficiently motivated and had ample opportunities to develop the target behaviours, none of the chimpanzees tested made or used flakes in any of the experimental conditions. Conclusions : These results differ from all previous ape flaking experiments, which found flake manufacture and use in bonobos and one orangutan. However, these earlier studies tested human-enculturated apes and provided test subjects with flake making and using demonstrations. The contrast between these earlier positive findings and our negative findings (despite using a much larger sample size) suggests that enculturation and/or demonstrations may be necessary for chimpanzees to acquire these abilities. The data obtained in this study are consistent with the hypothesis that flake manufacture and use might have evolved in the hominin lineage after the split between Homo and Pan 7 million years ago, a scenario further supported by the initial lack of flaked stone tools in the archaeological record after this split. We discuss possible evolutionary scenarios for flake manufacture and use in both non-hominin and hominin lineages., Competing Interests: No competing interests were disclosed., (Copyright: © 2021 Bandini E et al.)

Prostate Cancer (PCa) is one of the most frequently identified urological cancers. PCa patients are often over-diagnosed due to still not highly specific diagnostic methods. The need for more accurate diagnostic tools to prevent overestimated diagnosis and unnecessary treatment of patients with non-malignant conditions is clear, and new markers and methods are strongly desirable. Extracellular vesicles (EVs) hold great promises as liquid biopsy-based markers. Despite the biological and technical issues present in their detection and study, these particles can be found highly abundantly in the biofluid and encompass a wealth of macromolecules that have been reported to be related to many physiological and pathological processes, including cancer onset, metastasis spreading, and treatment resistance. The present study aims to perform a technical feasibility study to develop a new workflow for investigating EVs from several biological sources. Serum and urinary supernatant EVs of PCa, benign prostatic hyperplasia (BPH) patients, and healthy donors were isolated and investigated by a fast, easily performable, and cost-effective cytofluorimetric approach for a multiplex detection of 37 EV-antigens. We also observed significant alterations in serum and urinary supernatant EVs potentially related to BPH and PCa, suggesting a potential clinical application of this workflow.

Background: Myelofibrosis (MF) is a clonal disorder of hemopoietic stem/progenitor cells (HSPCs) with high prevalence in elderly patients and mutations in three driver genes (JAK2, MPL, or CALR). Around 10-15% of patients are triple-negative (TN) for the three driver mutations and display significantly worse survival. Circulating extracellular vesicles (EVs) play a role in intercellular signaling and are increased in inflammation and cancer. To identify a biomolecular signature of TN patients, we comparatively evaluated the circulating HSPCs and their functional interplay with the microenvironment focusing on EV analysis., Methods: Peripheral blood was collected from MF patients (n = 29; JAK2 V617F mutation, n = 23; TN, n = 6) and healthy donors (HD, n = 10). Immunomagnetically isolated CD34 + cells were characterized by gene expression profiling analysis (GEP), survival, migration, and clonogenic ability. EVs were purified from platelet-poor plasma by ultracentrifugation, quantified using the Nanosight technology and phenotypically characterized by flow cytometry together with microRNA expression. Migration and survival of CD34 + cells from patients were also analyzed after in vitro treatments with selected inflammatory factors, i.e. (Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α, IL6) or after co-culture with EVs from MF patients/HD., Results: The absolute numbers of circulating CD34 + cells were massively increased in TN patients. We found that TN CD34 + cells show in vitro defective functions and are unresponsive to the inflammatory microenvironment. Of note, the plasma levels of crucial inflammatory cytokines are mostly within the normal range in TN patients. Compared to JAK2 V617F -mutated patients, the GEP of TN CD34 + cells revealed distinct signatures in key pathways such as survival, cell adhesion, and inflammation. Importantly, we observed the presence of mitochondrial components within plasma EVs and a distinct phenotype in TN-derived EVs compared to the JAK2 V617F -mutated MF patients and HD counterparts. Notably, TN EVs promoted the survival of TN CD34 + cells. Along with a specific microRNA signature, the circulating EVs from TN patients are enriched with miR-361-5p., Conclusions: Distinct EV-driven signals from the microenvironment are capable to promote the TN malignant hemopoiesis and their further investigation paves the way toward novel therapeutic approaches for rare MF.

Liquid biopsy is gaining importance in the context of analysis of circulating subcellular components, such as exosomes and nucleic acids, and the investigation of biological fluids is increasing because they express features common to the tissue of origin. Particularly, urine has become one of the most attractive biofluids in clinical practice due to its easy collection approach, its availability of large quantities, and its noninvasiveness. Furthermore, a peculiarity is that, compared to serum or plasma, urine is characterized by a simpler composition that improves isolation and identification of biomarkers. Recent studies have been associated with the investigation of mRNAs and microRNAs as potential noninvasive cancer biomarkers in urine, and to date, several approaches for isolating and measuring urinary nucleic acids have been established, despite still developing. This chapter aims at giving some main published evidences on urinary microRNAs and mRNAs, with the intent to consider their potential translational use in clinical practice.

Studies on primate tool-use often involve the use of baseline conditions, as they allow for the examination of any differences in the subjects' behavior before and after the introduction of a tool-use task. While these baseline conditions can be powerful for identifying the relative contributions of individual and social learning for the acquisition of tool-use behaviors in naïve (usually captive) subjects, many have criticized them for being too short, and not allowing enough time for the behavior to develop spontaneously. Furthermore, some wild tool-use behaviors such as chimpanzee nut-cracking require animals to manipulate and familiarize themselves with the materials of the behavior within a "sensitive learning period" before it develops later on in life. One solution to this problem is to implement long-term baselines, in which, with collaboration with zoological institutions, the materials of the behavior are left in the enclosure for an extended period. The keepers would then be asked not to demonstrate or train the animals in the target behavior, but to report back to the researchers if they observe the behavior emerge during this extended period. Alongside keeper reports, video cameras could be installed in the enclosure to minimize the chance of false negatives and to allow for coding and inter-rater reliability to be carried out on the videos. These long-term baselines therefore provide extended enrichment opportunities for the animals, alongside allowing the zoological institution to publicize their involvement with the study and guests to observe animals interacting with different testing apparatuses and tools. Finally, long-term baselines can provide invaluable insight on the individual and social learning abilities of primates as well as the potential development stages and sensitive learning periods required for specific behaviors., (© 2020 The Authors. American Journal of Primatology published by Wiley Periodicals LLC.)

The critical examination of current hypotheses is one of the key ways in which scientific fields develop and grow. Therefore, any critique, including Haidle and Schlaudt's article, "Where Does Cumulative Culture Begin? A Plea for a Sociologically Informed Perspective," represents a welcome addition to the literature. However, critiques must also be evaluated. In their article, Haidle and Schlaudt (Biol Theory 15:161-174, 2020. 10.1007/s13752-020-00351-w; henceforth H&S) review some approaches to culture and cumulative culture in both human and nonhuman primates. H&S discuss the "zone of latent solutions" (ZLS) hypothesis as applied to nonhuman primates and stone-toolmaking premodern hominins. Here, we will evaluate whether H&S's critique addresses its target., (© The Author(s) 2021.)

The analysis of liquid biopsy as a source of diagnostic, prognostic, and predictive biomarkers is still object of the main research in the prostate cancer field. Many advantages, such as less invasiveness compared to plasma or serum analysis and the rich content, confer to urine a role as an interesting fluid to be analysed especially in urological diseases. Here we report a workflow focused on profile, concentration, and protein surface characterization of EVs from urinary supernatant.