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Abstract The hypothalamus contains a remarkable diversity of neurons that orchestrate behavioural and metabolic outputs in a highly plastic manner. Neuronal diversity is key to enabling hypothalamic functions and, according to the neuroscience dogma, it is predetermined during embryonic life. Here, by combining lineage tracing of hypothalamic pro-opiomelanocortin (Pomc) neurons with single-cell profiling approaches in adult male mice, we uncovered subpopulations of ‘Ghost’ neurons endowed with atypical molecular and functional identity. Compared to ‘classical’ Pomc neurons, Ghost neurons exhibit negligible Pomc expression and are ‘invisible’ to available neuroanatomical approaches and promoter-based reporter mice for studying Pomc biology. Ghost neuron numbers augment in diet-induced obese mice, independent of neurogenesis or cell death, but weight loss can reverse this shift. Our work challenges the notion of fixed, developmentally programmed neuronal identities in the mature hypothalamus and highlight the ability of specialised neurons to reversibly adapt their functional identity to adult-onset obesogenic stimuli.

Abstract Background Immune checkpoint inhibitors (ICIs), administered alone or combined with chemotherapy, are the standard of care in advanced non-oncogene addicted Non-Small Cell Lung Cancer (NSCLC). Despite these treatments' success, most long-term survival benefit is restricted to approximately 20% of patients, highlighting the need to identify novel biomarkers to optimize treatment strategies. In several solid tumors, immune soluble factors, the activatory CD137+ Tcells, and the immunosuppressive cell subsets Tregs and MDSCs (PMN(Lox1+)-MDSC and M-MDSCs) correlated with responses to ICIs and clinical outcomes thus becoming appealing predictive and prognostic factors. This study investigated the role of distinct CD137+ Tcell subsets, Tregs, MDSCs, and immune-soluble factors in NSCLC patients as possible biomarkers. Methods The levels of T cells, MDSCs and soluble factors were evaluated in 89 metastatic NSCLC patients who underwent ICIs as first- or second-line treatment. T cell analysis was performed by cytoflurimetry evaluating Tregs and different CD137+ Tcell subsets also combined with CD3+, CD8+, PD1+, and Ki67+ markers. Circulating cytokines and immune checkpoints were also evaluated by Luminex analysis. All these parameters were correlated with several clinical factors (age, sex, smoking status, PS and TPS), response to therapy, PFS , and OS . The analyses were conducted in the overall population and in patients treated with ICIs as first-line (naïve patients). Results In both groups of patients, high levels of circulating CD137+ and CD137+PD1+ T cells (total, CD4 and CD8) and the soluble factor LAG3 positively correlated with response to therapy. In naïve patients, PMN(Lox1+)-MDSCs negatively correlated with clinical response, and a high percentage of Tregs was associated with favorable survival. Moreover, the balance between Treg/CD137+ Tcells or PMN(Lox1+)-MDSC/CD137+ Tcells was higher in non-responding patients and was associated with poor survival. CD137+ Tcells and Tregs resulted as two positive independent prognostic factors. Conclusion High levels of CD137+, CD137+PD1+ Tcells and sLAG3 could predict the response to ICIs in NSCLC patients independently by previous therapy. Combining the evaluation of CD137+ Tcells and Tregs also as Treg/CD137+ T cells ratio it is possible to identify naive patients with longer survival.

Inkjet printing is considered a key technology in the fabrication of organic light-emitting diodes (OLEDs), but achieving a fully inkjet-printable OLED structure is still a challenge. Here, we propose the fabrication of OLEDs with an inkjet-printed multilayer structure (i.e. anode/hole injection layer (HIL)/electron blocking layer (EBL)/emitting layer (EML)) by properly developing new HIL and EBL inks to achieve uniform and homogeneous printed thin films. In the fabricated multilayer device, the dissolution process of the EBL, which occurs during the printing of the EML, creates a blurred interface, resulting in device performance that achieves maximum current efficiencies of 20 cd/A and 7 cd/A with ITO and printed polymeric anode, respectively. With the aim of simplifying the structure of the device and mimicking the formation of such a blurred interface, another printed multilayer structure (i.e. anode/printed HIL/printed EBL:EML) was proposed, achieving maximum current efficiencies of 13 and 6 cd/A with ITO and polymeric anode, respectively. Such results represent a compromise between simplifying the fabrication process and achieving good electro-optical properties and thus represent a further step towards the fabrication of a fully inkjet-printed ITO-free OLED.

Abstract Background Fibroblast growth factor receptor (FGFR) gene family alterations are found in several cancers, indicating their importance as potential therapeutic targets. The FGFR-tyrosine kinase inhibitor (TKI) pemigatinib has been introduced in the treatment of advanced cholangiocarcinoma and more recently for relapsed or refractory myeloid/lymphoid neoplasms with FGFR2 and FGFR1 rearrangements, respectively. Several clinical trials are currently investigating the possible combination of pemigatinib with immunotherapy. In this study, we analyzed the biological and molecular effects of pemigatinib on different cancer cell models (lung, bladder, and gastric), which are currently objective of clinical trial investigations. Methods NCI-H1581 lung, KATO III gastric and RT-112 bladder cancer cell lines were evaluated for FGFR expression by qRT-PCR and Western blot. Cell lines were treated with Pem and then characterized for cell proliferation, apoptosis, production of intracellular reactive oxygen species (ROS), and induction of senescence. The expression of microRNAs with tumor suppressor functions was analyzed by qRT-PCR, while modulation of the proteins coded by their target genes was evaluated by Western blot and mRNA. Descriptive statistics was used to analyze the various data and student’s t test to compare the analysis of two groups. Results Pemigatinib exposure triggered distinct signaling pathways and reduced the proliferative ability of all cancer cells, inducing G1 phase cell cycle arrest and strong intracellular stress resulting in ROS production, senescence and apoptosis. Pemigatinib treatment also caused the upregulation of microRNAs (miR-133b, miR-139, miR-186, miR-195) with tumor suppressor functions, along with the downregulation of validated protein targets with oncogenic roles (c-Myc, c-MET, CDK6, EGFR). Conclusions These results contribute to clarifying the biological effects and molecular mechanisms mediated by the anti-FGFR TKI pemigatinib in distinct tumor settings and support its exploitation for combined therapies.

Abstract The fabrication of optoelectronic devices using low‐cost inkjet printing techniques is a topic of great interest to the scientific and industrial community and represents a step toward the full deployment of solution‐processable organic light emitting diodes (OLEDs), particularly for commercial lighting and signaling applications. Herein, the inkjet printing of tBuCzDBA (9,10‐bis(4‐(3,6‐di‐tert‐butyl‐9H‐carbazol‐9‐yl)−2,6‐dimethylphenyl)−9,10‐diboraanthracene) is reported, a high‐performing thermally activated delayed fluorescence (TADF) emitter for OLEDs. Optimizing the surface tension values of the ink formulations and the associated wetting behavior are crucial parameters for achieving a uniform and homogeneous printed thin film. In particular, it is observed that using a proper mixture of solvents with different surface tensions, it is possible to generate Marangoni flows inside the drop, which triggers a very fast drying process, ensuring optimized morphological and optical properties in the inkjet printed tBuCzDBA‐based film. OLEDs exploiting this film as an emissive layer are then fabricated, achieving a maximum luminance of 32 000 cd m−2, a current efficiency of 27.5 cd A−1, and an external quantum efficiencyof 10%. To the best of the knowledge, this is the highest efficiency reported to date for self‐hosted TADF inkjet‐printed OLEDs.

The exposure to sublethally high temperature reduces reproductive performance in diverse organisms. Although this effect has been particularly emphasized for males or male reproductive functioning, it remains largely unknown whether the effect of heat on fertility is sex-specific. Here we examined the impact of sublethally high temperature on male and female functions in a simultaneously hermaphroditic snail species, Lymnaea stagnalis. Examining hermaphrodites is useful to evaluate the sex-specific impacts of heat exposure, since they possess male and female functions within a single individual, sharing genetic and environmental factors. Moreover, previously developed sex allocation theory allows us to compare the differential performance of sex functions. In this study, we exposed snails to 20°C (control), 24°C and 28°C for 14 days and assessed their egg and sperm production, sperm transfer, mating behaviour and growth. Both types of gamete production were significantly reduced by higher temperature, leading to an overall reduction of reproductive investment. By quantifying sex allocation, we furthermore revealed that the heat-stressed snails reduced the relative investment in their male function. This study illustrates that examining simultaneous hermaphrodites can provide significant insights for the impact of heat, and the proximate mechanism, on reproduction in diverse organisms.

Abstract Exposure to extreme temperatures can negatively affect animal reproduction, by disrupting the ability of individuals to produce any offspring (fertility), or the number of offspring produced by fertile individuals (fecundity). This has important ecological consequences, because reproduction is the ultimate measure of population fitness: a reduction in reproductive output lowers the population growth rate and increases the extinction risk. Despite this importance, there have been no large‐scale summaries of the evidence for effect of temperature on reproduction. We provide a systematic map of studies testing the relationship between temperature and animal reproduction. We systematically searched for published studies that statistically test for a direct link between temperature and animal reproduction, in terms of fertility, fecundity or indirect measures of reproductive potential (gamete and gonad traits). Overall, we collated a large and rich evidence base, with 1654 papers that met our inclusion criteria, encompassing 1191 species. The map revealed several important research gaps. Insects made up almost half of the dataset, but reptiles and amphibians were uncommon, as were non‐arthropod invertebrates. Fecundity was the most common reproductive trait examined, and relatively few studies measured fertility. It was uncommon for experimental studies to test exposure of different life stages, exposure to short‐term heat or cold shock, exposure to temperature fluctuations, or to independently assess male and female effects. Studies were most often published in journals focusing on entomology and pest control, ecology and evolution, aquaculture and fisheries science, and marine biology. Finally, while individuals were sampled from every continent, there was a strong sampling bias towards mid‐latitudes in the Northern Hemisphere, such that the tropics and polar regions are less well sampled. This map reveals a rich literature of studies testing the relationship between temperature and animal reproduction, but also uncovers substantial missing treatment of taxa, traits, and thermal regimes. This database will provide a valuable resource for future quantitative meta‐analyses, and direct future studies aiming to fill identified gaps.

Abstract The endocannabinoid system (ECS) regulates energy metabolism, has been implicated in the pathogenesis of metabolic diseases and exerts its actions mainly through the type 1 cannabinoid receptor (CB1). Likewise, autophagy is involved in several cellular processes. It is required for the normal development of muscle mass and metabolism, and its deregulation is associated with diseases. It is known that the CB1 regulates signaling pathways that control autophagy, however, it is currently unknown whether the ECS could regulate autophagy in the skeletal muscle of obese mice. This study aimed to investigate the role of the CB1 in regulating autophagy in skeletal muscle. We found concomitant deregulation in the ECS and autophagy markers in high-fat diet-induced obesity. In obese CB1-KO mice, the autophagy-associated protein LC3 II does not accumulate when mTOR and AMPK phosphorylation levels do not change. Acute inhibition of the CB1 with JD-5037 decreased LC3 II protein accumulation and autophagic flux. Our results suggest that the CB1 regulates autophagy in the tibialis anterior skeletal muscle in both lean and obese mice.

Microfluidics offers a highly controlled and reproducible route to synthesize lipid vesicles. In recent years, several microfluidic approaches have been introduced for this purpose, but double emulsions, such as Water-in-Oil-in-Water (W/O/W) droplets, are preferable to produce giant vesicles that are able to maximize material encapsulation. Flow focusing (FF) is a technique used to generate double emulsion droplets with high monodispersity, a controllable size, and good robustness. Many researchers use polydimethylsiloxane as a substrate material to fabricate microdroplet generators, but it has some limitations due to its hydrophobicity, incompatibility with organic solvents, and the molecular adsorption on the microchannel walls. Thus, specific surface modification and functionalization steps, which are uncomfortable to perform in closed microchannels, are required to overcome these shortcomings. Here, we propose glass as a material to produce a chip with a six-inlet junction geometry. The peculiar geometry and the glass physicochemical properties allow for W/O/W droplet formation without introducing microchannel wall functionalization and using a variety of reagents and organic solvents. The robust glass chip can be easily cleaned and used repeatedly, bringing advantages in terms of cost and reproducibility in emulsion preparation.

Membrane-based sensors (MePSs) exhibit remarkable precision and sensitivity in detecting pressure changes. MePSs are commonly used to monitor catalytic reactions in solution, generating gas products crucial for signal amplification in bioassays. They also allow for catalyst quantification by indirectly measuring the pressure generated by the gaseous products. This is particularly interesting for detecting enzymes in biofluids associated with disease onset. To enhance the performance of a MePS, various structural factors influence membrane flexibility and response time, ultimately dictating the device’s pressure sensitivity. In this study, we fabricated MePSs using polydimethylsiloxane (PDMS) and investigated how structural modifications affect the Young’s modulus (E) and residual stress (σ0) of the membranes. These modifications have a direct impact on the sensors’ sensitivity to pressure variations, observed as a function of the volume of the chamber (Σ) or of the mechanical properties of the membrane itself (S). MePSs exhibiting the highest sensitivities were then employed to detect catalyst quantities inducing the dismutation of hydrogen peroxide, producing dioxygen as a gaseous product. As a result, a catalase enzyme was successfully detected using these optimized MePSs, achieving a remarkable sensitivity of (22.7 ± 1.2) µm/nM and a limit of detection (LoD) of 396 pM.

BackgroundThe immune profile of each patient could be considered as a portrait of the fitness of his/her own immune system. The predictive role of the immune profile in immune-related toxicities (irAEs) development and tumour response to treatment was investigated.MethodsA prospective, multicenter study evaluating, through a multiplex assay, the soluble immune profile at the baseline of 53 patients with advanced cancer, treated with immunotherapy as single agent was performed. Four connectivity heat maps and networks were obtained by calculating the Spearman correlation coefficients for each group: responder patients who developed cumulative toxicity (R-T), responders who did not develop cumulative toxicity (R-NT), non-responders who developed cumulative toxicity (NR-T), non-responders who did not develop cumulative toxicity (NR-NT).ResultsA statistically significant up-regulation of IL-17A, sCTLA4, sCD80, I-CAM-1, sP-Selectin and sEselectin in NR-T was detected. A clear loss of connectivity of most of the soluble immune checkpoints and cytokines characterized the immune profile of patients with toxicity, while an inversion of the correlation for ICAM-1 and sP-selectin was observed in NR-T. Four connectivity networks were built for each group. The highest number of connections characterized the NR-T.ConclusionsA connectivity network of immune dysregulation was defined for each subgroup of patients, regardless of tumor type. In patients with the worst prognosis (NR-T) the peculiar connectivity model could facilitate their early and timely identification, as well as the design of a personalized treatment approach to improve outcomes or prevent irAEs.

Primary cutaneous CD30+ lymphoproliferative disorders include primary cutaneous anaplastic large cell lymphoma (pcALCL) and lymphomatoid papulosis. The prognosis of the disease is usually excellent but, in a minority of cases, it presents with extracutaneous involvement and aggressive behavior. The case we present—relapsed after surgical excision, immunosuppressive therapy, and conventional chemotherapy—is the first one treated with Autologous Stem Cell transplant followed by Brentuximab Vedotin consolidation, a scheme already used for high risk Hodgkin Lymphoma.

Polymeric membranes are useful tools for water filtration processes, with their performance strongly dependent on the presence of hydrophilic dopants. In this study, polyaniline (PANI)-capped aluminosilicate (halloysite) nanotubes (HNTs) are dispersed into polyether sulfone (PES), with concentrations ranging from 0.5 to 1.5 wt%, to modify the properties of the PES membrane. Both undoped and HNT-doped PES membranes are investigated in terms of wettability (static and time-dependent contact angle), permeance, mechanical resistance, and morphology (using scanning electron microscopy (SEM)). The higher water permeance observed for the PES membranes incorporating PANI-capped HNTs is, finally, assessed and discussed vis-à-vis the real distribution of HNTs. Indeed, the imaging and characterization in terms of composition, spatial arrangement, and counting of HNTs embedded within the polymeric matrix are demonstrated using non-destructive Micro Particle Induced X-ray Emission (µ-PIXE) and Scanning Transmission Ion Microscopy (STIM) techniques. This approach not only exhibits the unique ability to detect/highlight the distribution of HNTs incorporated throughout the whole thickness of polymer membranes and provide volumetric morphological information consistent with SEM imaging, but also overcomes the limits of the most common analytical techniques exploiting electron probes. These aspects are comprehensively discussed in terms of practical analysis advantages.

Objective. The role of T cells in the pathogenesis of systemic lupus erythematosus (SLE) has recently gained attention. Costimulatory molecules are membrane proteins strictly associated with T-cell receptor (TCR), acting by activating or inhibiting T cells and antigen-presenting cells (APC) through direct and reverse signaling, thus becoming responsible for the development of effector T cells or regulatory T cells. The primary objective of the present case–control study was to evaluate the cell membrane expression of CD137 on T cells and the serum concentration of CD137 (sCD137) in a SLE cohort. Materials. We enrolled SLE patients and sex/age-matched healthy subjects (HS). Disease activity was assessed by SLEDAI-2K. By application of flow cytometry, we evaluated the expression of CD137 on CD4+ and CD8+ lymphocytes. ELISA test was performed to evaluate serum levels of sCD137. Results. Twenty-one SLE patients (M/F 1/20; median age 48 years (IQR 17); median disease duration 144 months (IQR 204)) were evaluated. SLE patients showed %CD3+CD137+ cells significantly higher compared to HS (median 5.32 (IQR 6.11) versus 3.3 (IQR 1.8), p=0.001). In SLE patients, %CD4+CD137+ cells positively correlated with SLEDAI-2K (p=0.0082, r = 0.58, CI (0.15–0.82); indeed, %CD4+CD137+ cells were significantly lower in SLE patients with a remission status compared to those not reaching this condition (median 1.07 (IQR 0.91) versus 1.58 (IQR 2.42), p=0.013). Accordingly, sCD137 levels were significantly lower in remission status (31.30 pg/mL (IQR 102.2 versus median 122.8 pg/mL (IQR 536); p=0.03) and correlated with %CD4+CD137+ cells (p=0.012, r = 0.60, CI (0.15–0.84)). Conclusion. Our results suggest a possible involvement of CD137-CD137L axis in SLE pathogenesis, as demonstrated by higher expression of CD137 on CD4+ cells in SLE compared with HS. Furthermore, the positive correlation between SLEDAI-2K and membrane CD137 expression on CD4+ cells, as well as soluble CD137, indicates a possible use as biomarkers for disease activity.

BackgroundDespite the efficacy of immunotherapy, only a small percentage of patients achieves a long-term benefit in terms of overall survival. The aim of this study was to define an immune profile predicting the response to immune checkpoint inhibitors (ICIs).MethodsPatients with advanced solid tumors, who underwent ICI treatment were enrolled in this prospective study. Blood samples were collected at the baseline. Thirteen soluble immune checkpoints, 3 soluble adhesion molecules, 5 chemokines and 11 cytokines were analyzed. The results were associated with oncological outcomes.ResultsRegardless of tumor type, patients with values of sTIM3, IFNα, IFNγ, IL1β, IL1α, IL12p70, MIP1β, IL13, sCD28, sGITR, sPDL1, IL10 and TNFα below the median had longer overall survival (p

Pembrolizumab, an anti-PD-1 antibody, has been approved as first-line treatment for recurrent or metastatic head and neck squamous cell carcinoma ((R/M) HNSCC). However, only a minority of patients benefit from immunotherapy, which highlights the need to identify novel biomarkers to optimize treatment strategies. CD137+ T cells have been identified as tumour-specific T cells correlated with immunotherapy responses in several solid tumours. In this study, we investigated the role of circulating CD137+ T cells in (R/M) HNSCC patients undergoing pembrolizumab treatment. PBMCs obtained from 40 (R/M) HNSCC patients with a PD-L1 combined positive score (CPS) ≥1 were analysed at baseline via cytofluorimetry for the expression of CD137, and it was found that the percentage of CD3+CD137+ cells is correlated with the clinical benefit rate (CBR), PFS, and OS. The results show that levels of circulating CD137+ T cells are significantly higher in responder patients than in non-responders (p = 0.03). Moreover, patients with CD3+CD137+ percentage ≥1.65% had prolonged OS (p = 0.02) and PFS (p = 0.02). Multivariate analysis, on a combination of biological and clinical parameters, showed that high levels of CD3+CD137+ cells (≥1.65%) and performance status (PS) = 0 are independent prognostic factors of PFS (CD137+ T cells, p = 0.007; PS, p = 0.002) and OS (CD137+ T cells, p = 0.006; PS, p = 0.001). Our results suggest that levels of circulating CD137+ T cells could serve as biomarkers for predicting the response of (R/M) HNSCC patients to pembrolizumab treatment, thus contributing to the success of anti-cancer treatment.

Metastatic uveal melanoma (UM) is a poor prognosis malignancy. Immunotherapy is commonly employed, despite the low activity, considering the lack of other effective systemic treatments. In this study, the prognostic and predictive role of soluble immune checkpoints and inflammatory cytokines/chemokines in 22 metastatic UM patients was evaluated. Baseline levels of these molecules were assessed, as well as their changes during anti-PD-1 therapy. The correlation between soluble immune checkpoints/cytokines/chemokines and survival was analyzed. A comparison between circulating immune profile of metastatic cutaneous melanoma (CM), for which immunotherapy is a mainstay of treatment, and UM during anti-PD-1 therapy was also performed. Three immune molecules resulted significantly higher in metastatic UM patients with survival 30 months. We also observed an increase of sCD137, sCD28, sPD-1, sPD-L2 sLAG3, sCD80 and sTim3 during anti-PD-1 treatment, as well as IDO activity, IP-10 and CCL2. Several of these molecules were significantly higher in UM compared to CM patients during anti-PD-1 therapy. The analysis of circulating immune molecules allows to identify patients with poor prognosis despite immunotherapy and patients with long survival treated with an anti-PD-1 agent. The different serum concentration of these molecules during anti-PD-1 therapy between UM and CM reflects the different efficacy of immune checkpoint inhibitors.

Summary: Background: Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) are innovative small target molecules that, in combination with endocrine therapy, have recently been employed in the treatment of patients with HR+/HER2− metastatic breast cancer (mBC). In this prospective study, we investigate the impact of CDK4/6i on the immune profile of patients with HR+/HER2− mBC. Methods: Immune cell subsets were analysed using flow cytometry of peripheral blood mononuclear cells (PBMCs) isolated from patients with HR+/HER2− mBC, both before and during treatment. Regulatory T cells (Tregs) were identified using the markers CD4, CD25, CTLA4, CD45RA, and intracellular FOXP3. Monocytic and polymorphonuclear myeloid-derived suppressor cells (M-MDSCs and PMN-MDSCs) and other immune populations were analysed using CD45, CD14, CD66b, CD11c, HLA-DR, CD3, CD8, CD28, CD137, PD1, CD45RA, CCR7, and Ki67. Findings: The percentage of circulating Tregs and M/PMN-MDSCs was significantly downregulated from baseline during CDK4/6i-treatment (p

Highly conductive PEDOT:PSS is one of the most promising materials for indium tin oxide (ITO) substitution in printed electronics. Here, we report the development and optimisation of two PEDOT:PSS ink formulations for the fabrication of inkjet-printed transparent conductive layers. Starting from aqueous commercial solutions, co-solvents and a non-ionic surfactant were employed to modify the surface tension, improve the wetting capability of the ink, and obtain uniform and homogeneous thin films. In particular, the quantities of ethanol and surfactant were systematically adjusted to determine the optimal conditions for inkjet printing. The results demonstrate that a surface tension value between 28 and 40 mN/m and approximately 40 vol.% of a low-boiling-point co-solvent are fundamental to ensure the proper wetting of the glass substrate and a quick-drying process that confers uniformity to the printed thin film. The printed PEDOT:PSS thin films show good morphological, optical, and electrical properties that are similar to those observed for the corresponding spin-coated layers. The organic light-emitting diodes (OLEDs) fabricated with the inkjet-printed PEDOT:PSS electrodes showed a maximum quantum efficiency of 5.5% and a maximum current efficiency of 15 cd/A, which is comparable to spin-coated reference devices. Our study demonstrates the great potential of polymeric electrodes for the fabrication of high-efficiency printed OLED devices that are compatible with flexible and stretchable substrates.