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In the quest to find powerful modifiers of screen-printed electrodes for sensing applications, a set of rare earth-doped Ca10−xREx(PO4)6(OH)2 (RE = La, Nd, Sm, Eu, Dy, and Tm and x = 0.01, 0.02, 0.10, and 0.20) hydroxyapatite (HAp) samples were subjected to an in-depth electrochemical characterization using electrochemical impedance spectroscopy and cyclic and square wave voltammetry. Among all of these, the inorganic phosphates doped with lanthanum proved to be the most reliable, revealing robust analytical performances in terms of sensitivity, repeatability, reproducibility, and reusability, hence paving the way for their exploitation in sensing applications. Structural data on La-doped HAp samples were also provided by using different techniques, including optical microscopy, X-ray diffraction, Rietveld refinement from X-ray data, Fourier transform infrared, and Raman vibrational spectroscopies, to complement the electrochemical characterization.

Background and purpose While HCC is an inflammation-associated cancer, CRLM develops on permissive healthy liver microenvironment. To evaluate the immune aspects of these two different environments, peripheral blood-(PB), peritumoral-(PT) and tumoral tissues-(TT) from HCC and CRLM patients were evaluated. Methods 40 HCC and 34 CRLM were enrolled and freshly TT, PT and PB were collected at the surgery. PB-, PT- and TT-derived CD4+CD25+ Tregs, M/PMN-MDSC and PB-derived CD4+CD25− T-effector cells (Teffs) were isolated and characterized. Tregs’ function was also evaluated in the presence of the CXCR4 inhibitor, peptide-R29, AMD3100 or anti-PD1. RNA was extracted from PB/PT/TT tissues and tested for FOXP3, CXCL12, CXCR4, CCL5, IL-15, CXCL5, Arg-1, N-cad, Vim, CXCL8, TGFβ and VEGF-A expression. Results In HCC/CRLM-PB, higher number of functional Tregs, CD4+CD25hiFOXP3+ was detected, although PB-HCC Tregs exert a more suppressive function as compared to CRLM Tregs. In HCC/CRLM-TT, Tregs were highly represented with activated/ENTPD-1+Tregs prevalent in HCC. As compared to CRLM, HCC overexpressed CXCR4 and N-cadherin/vimentin in a contest rich in arginase and CCL5. Monocytic MDSCs were highly represented in HCC/CRLM, while high polymorphonuclear MDSCs were detected only in HCC. Interestingly, the function of CXCR4-PB-Tregs was impaired in HCC/CRLM by the CXCR4 inhibitor R29. Conclusion In HCC and CRLM, peripheral blood, peritumoral and tumoral tissues Tregs are highly represented and functional. Nevertheless, HCC displays a more immunosuppressive TME due to Tregs, MDSCs, intrinsic tumor features (CXCR4, CCL5, arginase) and the contest in which it develops. As CXCR4 is overexpressed in HCC/CRLM tumor/TME cells, CXCR4 inhibitors may be considered for double hit therapy in liver cancer patients.

Microgravity induced bone loss is a major concern for space travellers. Ground-based microgravity simulators are crucial to study the effect of microgravity exposure on biological systems and to address the limitations posed by restricted access to real space. In this work, for the first time we adopt a multidisciplinary approach to characterize the morphological, biochemical, and molecular changes underlying the response of human bone marrow stromal cells to long-term simulated microgravity exposure during osteogenic differentiation. Our results show that osteogenic differentiation is reduced while energy metabolism is promoted. We found novel proteins were dysregulated under simulated microgravity, including CSC1-like protein, involved in the mechanotransduction of pressure signals, and PTPN11, SLC44A1 and MME which are involved in osteoblast differentiation pathways and which may become the focus of future translational projects. The investigation of cell proteome highlighted how simulated microgravity affects a relatively low number of proteins compared to time and/or osteogenic factors and has allowed us to reconstruct a hypothetical pipeline for cell response to simulated microgravity. Further investigation focused on the application of nanomaterials may help to increase understanding of how to treat or minimalise the effects of microgravity.

Background and Purpose: Airway remodelling is a critical feature of chronic lung diseases. Epithelial-mesenchymal transition (EMT) represents an important source of myofibroblasts, contributing to airway remodelling. Here, we investigated the sphingosine-1-phosphate (S1P) role in EMT and its involvement in asthma-related airway dysfunction. Experimental Approach: A549 cells were used to assess the S1P effect on EMT and its interaction with TGF-β signalling. To assess the S1P role in vivo and its impact on lung function, two experimental models of asthma were used by exposing BALB/c mice to subcutaneous administration of either S1P or ovalbumin (OVA). Key Results: Following incubation with TGF-β or S1P, A549 acquire a fibroblast-like morphology associated with an increase of mesenchymal markers and down-regulation of the epithelial. These effects are reversed by treatment with the TGF-β receptor antagonist LY2109761. Systemic administration of S1P to BALB/c mice induces asthma-like disease characterized by mucous cell metaplasia and increased levels of TGF-β, IL-33 and FGF-2 within the lung. The bronchi harvested from S1P-treated mice display bronchial hyperresponsiveness associated with overexpression of the mesenchymal and fibrosis markers and reduction of the epithelial.The S1P-induced switch from the epithelial toward the mesenchymal pattern correlates to a significant increase of lung resistance and fibroblast activation. TGF-β blockade, in S1P-treated mice, abrogates these effects. Finally, inhibition of sphingosine kinases by SK1-II in OVA-sensitized mice, abrogates EMT, pulmonary TGF-β up-regulation, fibroblasts recruitment and airway hyperresponsiveness. Conclusion and Implications: Targeting S1P/TGF-β axis may hold promise as a feasible therapeutic target to control airway dysfunction in asthma. © 2021 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.

Intensive conventional agriculture and climate change have induced severe ecological damages and threatened global food security, claiming a reorientation of agricultural management and public policies towards a more sustainable development model. In this context, nanomaterials promise to support this transition by promoting mitigation, enhancing productivity, and reducing contamination. This review gathers recent research innovations on smart nanoformulations and delivery systems improving crop protection and plant nutrition, nanoremediation strategies for contaminated soils, nanosensors for plant health and food quality and safety monitoring, and nanomaterials as smart food-packaging. It also highlights the impact of engineered nanomaterials on soil microbial communities, and potential environmental risks, along with future research directions. Although large-scale production and in-field testing of nano-agrochemicals are still ongoing, the collected information indicates improvements in uptake, use efficiency, targeted delivery of the active ingredients, and reduction of leaching and pollution. Nanoremediation seems to have a low negative impact on microbial communities while promoting biodiversity. Nanosensors enable high-resolution crop monitoring and sustainable management of the resources, while nano-packaging confers catalytic, antimicrobial, and barrier properties, preserving food safety and preventing food waste. Though, the application of nanomaterials to the agri-food sector requires a specific risk assessment supporting proper regulations and public acceptance.

Simple Summary The management of advanced colorectal cancer (CRC) has been greatly improved with integrated strategies including stereotactic radiation therapy (SRT). It is a safe and effective option, particularly in oligo-metastatic (om) CRC patients. Interestingly, it has been demonstrated that SRT can induce regression of tumors in non-irradiated regions (“abscopal effect”) through stimulation of anti-tumor immune effects (“radiation-induced immunity”). We have recently shown that lung-limited omCRC is characterized by regression of tumor clones bearing specific key driver gene mutations. The aim of the PRELUDE-1 study is to assess the genetic and immunologic evolutions on tumor cancer/host cells induced by SRT in lung-limited omCRC through liquid biopsies and Next Generation Sequencing of tumor exome, HLA repertoire assessment, peripheral immune cells, and cytokine dynamics characterizations. An important secondary objective is the first prospective evaluation of the abscopal effect. The PRELUDE-1 results will help to identify subsets of patients more prone to show the abscopal effect. The PRELUDE-1 trial was registered into the clinicaltrials.gov registry on 22 April 2021, with identifier NCT04854213. Abstract Background: in recent years, the management of advanced colorectal cancer (CRC) has been greatly improved with integrated strategies including stereotactic radiation therapy (SRT). The administration of SRT has been demonstrated, particularly in oligo-metastatic (om) CRC, to be a safe and effective option. Interestingly, it has been demonstrated that SRT can induce regression of tumors in non-irradiated regions (“abscopal effect”) through stimulation of anti-tumor immune effects (“radiation-induced immunity”). We have recently shown that lung-limited omCRC is characterized by regression of tumor clones bearing specific key driver gene mutations. Aims: to assess the genetic evolution on tumor cancer cells induced by SRT in lung-limited omCRC. Secondary objectives included descriptions of the abscopal effect, responses’ duration, toxicity, and progression-free survival. A translational research will be performed to evaluate tumor genetic evolution (through liquid biopsies and Next Generation Sequencing), HLA class I repertoire, peripheral immune cells, and cytokine dynamics. Methods: PRELUDE-1 is a prospective translational study. SRT will be administered only to the largest nodule (with a maximum diameter ≤ 25 mm) in omCRC with two or three radiologically evident lesions. The sample size is based on the innovative hypothesis that radiation-induced immunity could induce regression of tumor clones bearing KRAS oncogene mutations. According to the binomial test, considering the frequency of KRAS mutations and assuming a probability of mutant KRAS→wild type KRAS of p0 = 0.0077, with α = 0.05 and 1-β = 0.60, the final sample size is 25 patients.

Background Inefficient T-cell access to the tumor microenvironment (TME) is among the causes of tumor immune-resistance. Previous evidence demonstrated that targeting CXCR4 improves anti-PD-1/PD-L1 efficacy reshaping TME. To evaluate the role of newly developed CXCR4 antagonists (PCT/IB2011/000120/ EP2528936B1/US2013/0079292A1) in potentiating anti-PD-1 efficacy two syngeneic murine models, the MC38 colon cancer and the B16 melanoma-human CXCR4-transduced, were employed. Methods Mice were subcutaneously injected with MC38 (1 × 106) or B16-hCXCR4 (5 × 105). After two weeks, tumors bearing mice were intraperitoneally (ip) treated with murine anti-PD-1 [RMP1–14] (5 mg/kg, twice week for 2 weeks), Pep R (2 mg/kg, 5 days per week for 2 weeks), or both agents. The TME was evaluated through immunohistochemistry and flow-cytometry. In addition, the effects of the human-anti-PD-1 nivolumab and/or Peptide-R54 (Pep R54), were evaluated on human melanoma PES43 cells and xenografts treated. Results The combined treatment, Pep R plus anti-PD-1, reduced the MC38 Relative Tumor Volume (RTV) by 2.67 fold (p = 0.038) while nor anti-PD-1, neither Pep R significantly impacted on tumor growth. Significant higher number of Granzyme B (GZMB) positive cells was detected in MC38 tumors from mice treated with the combined treatment (p = 0.016) while anti-PD-1 determined a modest but significant increase of tumor-infiltrating GZMB positive cells (p = 0.035). Also, a lower number of FoxP3 positive cells was detected (p = 0.022). In the B16-hCXCR4 tumors, two weeks of combined treatment reduced tumor volume by 2.27 fold while nor anti-PD-1 neither Pep R significantly impacted on tumor growth. A significant higher number of GRZB positive cells was observed in B16-hCXCR4 tumors treated with combined treatment (p = 0,0015) as compared to anti-PD-1 (p = 0.028). The combined treatment reduced CXCR4, CXCL12 and PD-L1 expression in MC38 tumors. In addition, flow cytometry on fresh B16-hCXCR4 tumors showed significantly higher Tregs number following anti-PD-1 partially reversed by the combined treatment Pep R and anti-PD-1. Combined treatment determined an increase of CD8/Tregs and CD8/MDSC ratio. To dissect the effect of anti-PD-1 and CXCR4 targeting on PD-1 expressed by human cancer cells, PES43 human melanoma xenograft model was employed. In vitro human anti-PD-1 nivolumab or pembrolizumab (10 μM) reduced PES43 cells growth while nivolumab (10 μM) inhibited pERK1/2, P38 MAPK, pAKT and p4EBP. PES43 xenograft mice were treated with Pep R54, a newly developed Pep R derivative (AcHN-Arg-Ala-[DCys-Arg- Nal(2′)-His-Pen]- COOH), plus nivolumab. After 3 weeks of combined treatment a significant reduction in tumor growth was shown (p = 0.038). PES43 lung disseminated tumor cells (DTC) were detected in fresh lung tissues as melanoma positive MCSP-APC+ cells. Although not statistically significant, DTC-PES43 cells were reduced in mice lungs treated with combined treatment while nivolumab or Pep R54 did not affect DTC number. Conclusion Combined treatment with the new developed CXCR4 antagonist, Pep R, plus anti-PD-1, reduced tumor-growth in two syngeneic murine models, anti-PD-1 sensitive and resistant, potentiating Granzyme and reducing Foxp3 cells infiltration. In addition, the human specific CXCR4 antagonist, Pep R54, cooperated with nivolumab in inhibiting the growth of the PD-1 expressing human PES43 melanoma xenograft. This evidence sheds light on PD-1 targeting mechanisms and paves the way for CXCR4/PD-1 targeting combination therapy.

Background: Hepatocellular carcinoma (HCC) and colorectal cancer liver metastasis (CRLM) represent the two most occurring liver cancer. Aim of the project was to analyze the impact of TME on HCC versus CRLM. In particular, Tregs were evaluated in tumor (TT), peritumoral (PT) tissue and peripheral blood (PB) of liver tumor to identify immune mechanisms and therapeutic targets. Methods: Tregs were characterized on PB/PT/TT cell suspension from 40 HCC and 34 CRLM by flow cytometry (FOXP3, CD25, CD4, CD45RA, CTLA-4, CXCR4, PD-1, ICOS and ENTPD1). PB-, PT- and TT-Tregs were isolated by Dynabeads Regulatory T-cell kit and tested for the capability to inhibit CFSE-labeled autologous T effector (Teff) proliferation. Tregs were also treated with Pep R29, a new CXCR4 antagonists, before coculturing with Teffs. Quantitative real-time PCR was performed to assess the expression of cyto-chemokines in PT and TT samples (IL-15, Arginase, CCL5, CXCL12). Results: In HCC/CRLM-PB higher number of CXCR4 (p Conclusions: These data suggest a more suppressive TME in HCC compared to CRLM and highlighted CXCR4 antagonism as a potential strategy to overcome Tregs-mediated TME immunosuppressive function in HCC and CRLM patients. Citation Format: Daniela Castaldo, Sara Santagata, Giuseppina Rea, Maria Napolitano, Crescenzo D'Alterio, Rita Guarino, Vittorio Albino, Andrea Belli, Carmen Cutolo, Francesco Izzo, Stefania Scala. Tumor microenvironment (TME) is more suppressive in hepatocellular carcinoma (HCC) than in colorectal cancer liver metastasis (CRLM): CXCR4 antagonism as strategy to revert T regulatory cells (Tregs) suppressive activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2550.

Herein, we describe three patients affected by metastatic colorectal cancer (mCRC) experiencing infection by severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) and reduction of disease burden during coronavirus disease 2019 (COVID-19) course. Insights into tumor-associated angiotensin-converting enzyme (ACE)-2 expression and lymphocyte function suggest a correlation between host/SARS-Cov-2 infection and tumor burden reduction. This may shed new light into (a) the infection mechanism of SARS-CoV-2 virus and (b) the multiple aspects of a composite antiviral immune response with potential paradoxical and unexpected applications.

In this paper, we provide an in-depth electrochemical characterization of a label-free impedimetric immunosensor for rapid detection of ochratoxin A. The sensor was based on a carbodiimide-mediated amide coupling reaction to immobilize a specific ochratoxin A antibody onto 4-mercaptobenzoic acid-modified commercial screen-printed gold electrode. Different variables affecting the performance of the developed sensor were optimized. Cyclic voltammetry and electrochemical impedance spectroscopy were used to analyse modifications of the interfacial properties occurring at each step of the biosensor assembly. The free electrode surface area, the diffusion coefficient, the peak-to-peak separation, the heterogeneous electron transfer constant, and charge transfer resistance have been calculated and compared. The decrease of charge transfer resistance values was linearly proportional to the ochratoxin A concentration in the range of 0.37– 2.86 ng/mL, with a detection limit of 0.19 ng/mL, a limit of quantification of 0.40 ng/mL, very good selectivity, reproducibility, and storage stability in the absence of antifouling agents. Surface morphology and topographic data at each step of the immunosensor assembly were studied by Atomic Force Microscopy, which also provided information on the specific binding of ochratoxin A. Finally, contact angle measurements revealed the hydrophilicity evolution of the surface during sensor assembly enabling OTA binding.

BackgroundColorectal cancer (CRC) is one of the most prevalent and deadly tumors worldwide. The majority of CRC is resistant to anti-programmed cell death-1 (PD-1)-based cancer immunotherapy, with approximately 15% with high-microsatellite instability, high tumor mutation burden, and intratumoral lymphocytic infiltration. Programmed death-ligand 1 (PD-L1)/PD-1 signaling was described in solid tumor cells. In melanoma, liver, and thyroid cancer cells, intrinsic PD-1 signaling activates oncogenic functions, while in lung cancer cells, it has a tumor suppressor effect. Our work aimed to evaluate the effects of the anti-PD-1 nivolumab (NIVO) on CRC cells.MethodsIn vitro NIVO-treated human colon cancer cells (HT29, HCT116, and LoVo) were evaluated for cell growth, chemo/radiotherapeutic sensitivity, apoptosis, and spheroid growth. Total RNA-seq was assessed in 6–24 hours NIVO-treated human colon cancer cells HT29 and HCT116 as compared with NIVO-treated PES43 human melanoma cells. In vivo mice carrying HT29 xenograft were intraperitoneally treated with NIVO, OXA (oxaliplatin), and NIVO+OXA, and the tumors were characterized for growth, apoptosis, and pERK1/2/pP38. Forty-eight human primary colon cancers were evaluated for PD-1 expression through immunohistochemistry.ResultsIn PD-1+ human colon cancer cells, intrinsic PD-1 signaling significantly decreased proliferation and promoted apoptosis. On the contrary, NIVO promoted proliferation, reduced apoptosis, and protected PD-1+ cells from chemo/radiotherapy. Transcriptional profile of NIVO-treated HT29 and HCT116 human colon cancer cells revealed downregulation of BATF2, DRAM1, FXYD3, IFIT3, MT-TN, and TNFRSF11A, and upregulation of CLK1, DCAF13, DNAJC2, MTHFD1L, PRPF3, PSMD7, and SCFD1; the opposite regulation was described in NIVO-treated human melanoma PES43 cells. Differentially expressed genes (DEGs) were significantly enriched for interferon pathway, innate immune, cytokine-mediated signaling pathways. In vivo, NIVO promoted HT29 tumor growth, thus reducing OXA efficacy as revealed through significant Ki-67 increase, pERK1/2 and pP38 increase, and apoptotic cell reduction. Eleven out of 48 primary human colon cancer biopsies expressed PD-1 (22.9%). PD-1 expression is significantly associated with lower pT stage.ConclusionsIn PD-1+ human colon cancer cells, NIVO activates tumor survival pathways and could protect tumor cells from conventional therapies.

Octreotide long-acting repeatable (LAR) is largely used to treat functional and/or metastatic neuroendocrine neoplasms (NENs). Its effect in controlling carcinoid syndrome and partially reduce tumour burden is attributable to the ability of octreotide to bind somatostatin receptors (SSTRs) on the tumour and metastasis, regulating growth hormone secretion and cell growth. Notably, SSTRs are also expressed, at different levels, on Tregs. Tregs, together with myeloid-derived suppressor cells (MDSCs), are key components in the anti-tumour immunoregulation. This is the first prospective study aimed to explore the impact of Octreotide (OCT) LAR on the immune system, with a particular focus on Tregs and MDSC cells. Here, we show that circulating Tregs are elevated in NENs patients compared to healthy donors and that treatment with OCT LAR significantly decrease the level of total Tregs and of the three functional Tregs populations: nTregs, eTregs and non-Tregs. Furthermore, OCT LAR treatment induces a functional impairment of the remaining circulating Tregs, significantly decreasing the expression of PD1, CTLA4 and ENTPD1. A trend in circulating MDSC cells is reported in patients treated with OCT LAR. The results reported here suggest that the effect of OCT LAR on Tregs could tip the balance of the patients&rsquo, immune-system towards a durable anti-tumour immunosurveillance with consequent long-term control of the NENs disease.

The chemokine receptor CXCR4 is overexpressed and functional in colorectal cancer. To investigate the role of CXCR4 antagonism in potentiating colon cancer standard therapy, the new peptide CXCR4 antagonist Peptide R (Pep R) was employed. Human colon cancer HCT116 xenograft-bearing mice were treated with chemotherapeutic agents (CT) 5-Fluorouracil (5FU) and oxaliplatin (OX) or 5FU and radio chemotherapy (RT-CT) in the presence of Pep R. After two weeks, CT plus Pep R reduced by 4-fold the relative tumor volume (RTV) as compared to 2- and 1.6-fold reductions induced, respectively, by CT and Pep R. In vitro Pep R addition to CT/RT-CT impaired HCT116 cell growth and further reduced HCT116 and HT29 clonal capability. Thus, the hypothesis that Pep R could target the epithelial mesenchyme transition (EMT) process was evaluated. While CT decreased ECAD and increased ZEB-1 and CD90 expression, the addition of Pep R restored the pretreatment expression. In HCT116 and HT29 cells, CT/RT-CT induced a population of CD133+CXCR4+ cells, supposedly a stem-resistant cancer cell population, while Pep R reduced it. Taken together, the results showed that targeting CXCR4 ameliorates the effect of treatment in colon cancer through inhibition of cell growth and reversal of EMT treatment-induced markers, supporting further clinical studies.

Hyperglycemia, obesity and metabolic syndrome are negative prognostic factors in breast cancer patients. Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment, achieving unprecedented efficacy in multiple malignancies. However, ICIs are associated with immune-related adverse events involving cardiotoxicity. We aimed to study if hyperglycemia could affect ipilimumab-induced anticancer efficacy and enhance its cardiotoxicity. Human cardiomyocytes and estrogen-responsive and triple-negative breast cancer cells (MCF-7 and MDA-MB-231 cell lines) were exposed to ipilimumab under high glucose (25 mM), low glucose (5.5 mM), high glucose and co-administration of SGLT-2 inhibitor (empagliflozin), shifting from high glucose to low glucose. Study of cell viability and the expression of new putative biomarkers of cardiotoxicity and resistance to ICIs (NLRP3, MyD88, cytokines) were quantified through ELISA (Cayman Chemical) methods. Hyperglycemia during treatment with ipilimumab increased cardiotoxicity and reduced mortality of breast cancer cells in a manner that is sensitive to NLRP3. Notably, treatment with ipilimumab and empagliflozin under high glucose or shifting from high glucose to low glucose reduced significantly the magnitude of the effects, increasing responsiveness to ipilimumab and reducing cardiotoxicity. To our knowledge, this is the first evidence that hyperglycemia exacerbates ipilimumab-induced cardiotoxicity and decreases its anticancer efficacy in MCF-7 and MDA-MB-231 cells. This study sets the stage for further tests on other breast cancer cell lines and primary cardiomyocytes and for preclinical trials in mice aimed to decrease glucose through nutritional interventions or administration of gliflozines during treatment with ipilimumab.

Carbon nanotubes (CNTs) are among the most exploited carbon allotropes in the emerging technologies of molecular sensing and bioengineering. However, the advancement of algal nanobiotechnology and nanobionics is hindered by the lack of methods for the straightforward visualization of the CNTs inside the cell. Herein, we present a handy and label-free experimental strategy based on visible Raman microscopy to assess the internalization of single-walled carbon nanotubes (SWCNTs) using the model photosynthetic alga Chlamydomonas reinhardtii as a recipient. The relationship between the properties of SWCNTs and their biological behavior was demonstrated, along with the occurrence of excitation energy transfer from the excited chlorophyll molecules to the SWCNTs. The non-radiative deactivation of the chlorophyll excitation promoted by the SWCNTs enables the recording of Raman signals originating from cellular compounds located near the nanotubes, such as carotenoids, polyphosphates, and starch. Furthermore, the outcome of this study unveils the possibility to exploit SWCNTs as spectroscopic probes in photosynthetic and non-photosynthetic systems where the fluorescence background hinders the acquisition of Raman scattering signals.

Here we investigatethe structural and biological effects ensuing form the disulfide bond replacement of a potent and selective C-X-C chemokine receptor type 4 (CXCR4) peptide antagonist, with 1,4- and 1,5- disubstituted 1,2,3-triazole moieties. Both strategies produced candidates that showed high affinity and selectivity against CXCR4. Notably, when assessed for their ability to modulate the CXCL12-mediated cell migration, the 1,4-triazole variant conserved the antagonistic effect in the low-mid nanomolar range, while the 1,5-triazole one displayed the ability to activate the migration, becoming the first in class low-molecular-weight CXCR4 peptide agonist. By combining NMR and computational studies, we provided a valuable model that highlighted differences in the interactions of the two peptidomimetics with the receptor that could account for their different functional profile. Finally, we envisage that our findings could be translated to different GPCR-interacting peptides for the pursuit of novel chemical probes that could assist in dissecting the complex puzzle of this fundamental class of transmembrane receptors.

Frequent occurrence of food contamination, growing concern about public safety, and the adoption of more stringent legislations have promoted research efforts in the agrifood industry as well as in environmental monitoring, attempting to guarantee improvements in food quality and safety. In addition, the increasing demand for sustainable agriculture has led to the introduction of novel processing methods for raw materials to achieve stable foods. For this reason, it is imperative that effiffi cient and reliable diagnostic tools be developed to detect potential h azards in agriculture and for food safety. In this context, biosensor technology represents the cutting-edge frontier in the agrifood and environmental fifields. Indeed, smart monitoring of chemical contaminants (i.e., pesticides, heavy metals, toxins, allergenic compounds, and food additives) represents one of the evolving issues challenging the assessment of food quality and safety. Progress in multianalyte screening, functional materials, microflfluidics and nanotechnology is amplifying the potential market of biosensors, making this technology more competitive in comparison with conventional lab-based procedures in terms of speed of response and operational fifield usage. However, despite the large number of publications and patents on biosensors for food analysis, only a few systems have been able to reach the market. Among the main drawbacks to be faced are the limited lifetime of biological components, mass production, as well as portability. In this chapter, current and future trends in analytical diagnostic tools are discussed, with the aim of promoting a deeper cooperation between research and industries and encouraging the use of biosensing technology as a more attractive option for the commercial sector.

Recent advances in nanotechnology applied to medicine and regenerative medicine have an enormous and unexploited potential for future space and terrestrial medical applications. The Nanoparticles and Osteoporosis (NATO) project aimed to develop innovative countermeasures for secondary osteoporosis affecting astronauts after prolonged periods in space microgravity. Calcium- and Strontium-containing hydroxyapatite nanoparticles (nCa-HAP and nSr-HAP, respectively) were previously developed and chemically characterized. This study constitutes the first investigation of the effect of the exogenous addition of nCa-HAP and nSr-HAP on bone remodeling in gravity (1 g), Random Positioning Machine (RPM) and onboard International Space Station (ISS) using human bone marrow mesenchymal stem cells (hBMMSCs). In 1 g conditions, nSr-HAP accelerated and improved the commitment of cells to differentiate towards osteoblasts, as shown by the augmented alkaline phosphatase (ALP) activity and the up-regulation of the expression of bone marker genes, supporting the increased extracellular bone matrix deposition and mineralization. The nSr-HAP treatment exerted a protective effect on the microgravity-induced reduction of ALP activity in RPM samples, and a promoting effect on the deposition of hydroxyapatite crystals in either ISS or 1 g samples. The results indicate the exogenous addition of nSr-HAP could be potentially used to deliver Sr to bone tissue and promote its regeneration, as component of bone substitute synthetic materials and additive for pharmaceutical preparation or food supplementary for systemic distribution.

Photosynthesis is receiving renewed interest due to the possibility to integrate whole plant cells or their photosynthetic active sub-components into optoelectronic devices such as biosensors and organic semiconductors. Photosynthetic assemblies use light energy to power electron transfer and charge separation across a charge-impermeable lipid membrane. One of the main working photosynthetic units is the pigment/protein complex photosystem II (PSII) which hosts, among others, the D1/D2 reaction center (RC) proteins, where most of the photosynthetic redox active components are located. Photonic energy is captured by the antenna systems and sequentially transferred towards the RC at the primary acceptor plastoquinone QA, a single-electron carrier tightly bound to the D2 protein. Thereafter, electrons are transferred from QA to the secondary plastoquinone QB, bound to the D1 subunit, via a non-heme iron. QB is a two-electron carrier and after full reduction and protonation to QBH2, it is displaced by another molecule of the plastoquinone pool [1]. Some classes of environmental pollutants are competitive inhibitors of QB binding and can interrupt the photosynthetic electron transport chain. This activity can be easily monitored and it is the working principle underlying the functioning of PSII-based biosensors for the detection of toxic compounds [2-4]. The primary goal of our studies is to determine how structural, dynamics and functional proprieties of natural and mutated photosynthetic D1 RC proteins in green algae influence the sequential electron transfer reactions leading to efficient photochemical energy conversion. A better understanding of these factors will help to identify the parameters underlying an increased performance in terms of protein stability and functional reliability for biosensoristic purposes and to design molecular systems mimicking the high efficiency of solar energy conversion in natural photosynthesis. While the structure and the function of those systems are known, the protein dynamics and its relationship with the activity is still a focus of interest. The understanding and the ability to modulate the existing relations between structure-dynamics-functionality through selective genetic mutation, is extremely important for the biotechnological applications and fundamental research studies. Recently, we measured the flexibility and diffusive dynamics of Chlamydomonas cells and thylakoids carrying both native and single point mutated D1 proteins using neutron spectroscopy. In particular, we analyzed a set of mutants having improved or reduced affinity for specific classes of herbicides, and increased tolerance to ionizing radiation [5-7] (Figure 1A). By combining elastic and quasi-elastic neutron scattering data with chlorophyll fluorescence measurements, we revealed that single aminoacid replacements in the D1- plastoquinone binding niche impair electron transfer efficiency, and notably affect the temperature dependence of the overall protein dynamics, inferring increased flexibility to the host membranes, expanding to the entire cells [8]. Furthermore, to get a more detailed dynamical picture and to better understand the relation with the activity of the photosynthetic RC, we completed the single particle dynamics experiment with a collective dynamics study by neutron Brillouin spectroscopy [9]. Results indicated that single point mutations affect collective density fluctuations in hydrating water providing important insight on the transmission of information possibly correlated to biological functionality (Figure 2). [1] J. M. Erickson et al., The Plant Cell, 1, 1989, 361-371. [2] I. Husu et al., Sens. Actuators. B Chem. 185, 2013, 321-330. [3] D.J. Swainsbury et al., Biosens. Bioelectron. 58, 2014, 172-178. [4] M. Chatzipetrou, et al., Electrochem. commun. 64, 2016, 46-50. [5] G. Rea et al., PLoS ONE 6, 2011, e16216. [6] M.D. Lambreva et al., PLoS ONE 8, 2013, e61851 [7] M Turemis et al., Sens. Actuators. B Chem. 241, 2017, 993-1001. [8] D. Russo et al., Biophys J, in press. [9] D. Russo et al., J. Phys. Chem. Lett. 7, 2016, 2429-2433. This work was supported by COST Action TD1102 and the Lazio Regional project n. 85-2017-15256.

Development of distant metastasis relies on interactions between cancer and stromal cells. CXCL12, also known as stromal-derived factor 1α (SDF-1α), is a major chemokine constitutively secreted in bone marrow, lymph nodes, liver and lung, playing a critical role in the migration and seeding of neoplastic cells. CXCL12 activates the CXCR4 receptor that is overexpressed in several human cancer cells. Recent evidence reveals that tumors induce pre-metastatic niches in target organ producing tumor-derived factors. Pre-metastatic niches represent a tumor growth-favoring microenvironment in absence of cancer cells. A commercially available dermal filler, hyaluronic acid (HA) -based gel, loaded with CXCL12 (CLG) reproduced a “fake” pre-metastatic niche. In vitro, B16-hCXCR4-GFP, human cxcr4 expressing murine melanoma cells efficiently migrated toward CLG. In vivo, CLGs and empty gels (EGs) were subcutaneously injected into C57BL/6 mice and 5 days later B16-hCXCR4-GFP cells were intravenously inoculated. CLGs were able to recruit a significantly higher number of B16-hCXCR4-GFP cells as compared to EGs, with reduced lung metastasis in mice carrying CLG. CLG were infiltrated by higher number of CD45-positive leukocytes, mainly neutrophils CD11b+Ly6G+ cells, myeloid CD11b+Ly6G- and macrophages F4/80. CLG recovered cells recapitulated the features of B16-hCXCR4-GFP (epithelial, melanin rich, MELAN A/ S100/ c-Kit/CXCR4 pos; α-SMA neg). Thus a HA-based dermal filler loaded with CXCL12 can attract and trap CXCR4+tumor cells. The CLG trapped cells can be recovered and biologically characterized. As a corollary, a reduction in CXCR4 dependent lung metastasis was detected.

Aim: Tumor cells can prime distant microenvironment building a pre-metastatic niche (PMN) where circulating tumor cells (CTCs) will home. 0.01% of CTCs, endowed with the capability to extravasate and infiltrate distant sites, will develop distant metastasis. In this process, a crucial role is played by chemokine gradients. Highly tumorigenic CTCs express the chemokine receptor CXCR4. CXCR4 ligand, CXCL12, attracts bone marrow-derived cells and CXCR4+ cancer cells in pre-metastatic sites to form a PMN. A CXCL12-loaded-hydrogel (CLG) was developed with the commercially available Belotero Hyaluronic gel dermal filler to develop a “pseudo niche” attracting immune and CTCs-CXCR4+cells. Aims: 1. Biological characterization of CTCs, 2. CTC/tumor microenvironment interplay 3. feasibility for clinical CTC isolation and characterization. Materials and methods: Human renal cancer cells SN12C (100 and 500) and A498 (500 and 1000) were seeded on 150ul Empty Gels (EG) or CLG [300ng/ml CXCL12], and allowed to migrate within gels, then fixed and DAPI-stained for enumeration or recovered by gel digestion and grown in complete media for 6 days. CXCR4 expression and colonies formation capability were evaluated. 100 SN12C or HT29, human colon cancer cells, were spiked in 7cc HD blood, ficoll-paqued and the mixture seeded on CLG. Isolated cells were fixed and stained with anti-hCD45-FITC, panCK-594 and DAPI (cancer cells identified as DAPI+/pan-CK+/CD45-). 7cc blood derived from 15 metastatic cancer patient’s (colon, lung, ovary, endometrial, kidney and glioblastoma) will be processed as previously described (CTCs identified as DAPI+/pan-CK+/CD45-); as comparison, CTCs will be isolated and enumerated using the commercially available Screen Cell™ filters according to manufacturer’s instructions. Results: A498 and SN12C cells efficiently infiltrate CLG as compared to empty gel (EG) (CLG/EG fold increase 1.6-1.9). SN12C and HT29 EG/CLG-recovered cells developed colonies after 6 days of culture. HT29-CLG recovered cells overexpressed CXCR4 compared to plastic grown cells and to EG cells and originates a higher number of colonies compared to plastic grown cells (171±21 for CLG VS 131±8 for plastic grown). SN12C and HT29 cells were successfully recovered in 7cc HD blood (recovery rate 20 and 14% respectively). Clinical trial on feasibility in isolating CTC cells on CLG recently started patient’s recruitment in ovarian, colon, lung and renal cancer metastatic patients. Conclusion: CLG-device mimics a PMN for capturing CTCs able to extravasate and infiltrate distant tissue, and potentially, more metastatic. CLG-isolated cells highly express CXCR4, develop higher number of colonies and migrate toward CXCL12. Moreover, the device allows cancer cell recovery from HD blood being potentially clinically useful for CTCs identification, characterization and trapping in cancer patients. Citation Format: Luigi Portella, Caterina Ieranò, Giulia Bertolini, Crescenzo D'Alterio, Giuseppina Rea, Sara Santagata, Anna Maria Trotta, Gelsomina Monaco, Roberto Pacelli, Stefania Scala. CXCL12 embedded-Hyaluronate based 'pseudo niche': a new device for CTCs/DTCs capturing and characterization [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO036.

Background: Regulatory T cells (Tregs) are responsible for maintaining self-tolerance, and inhibit antitumor immune responses. PD-1 blockade was reported to increase resistance of effector T cells to Tregs suppression and directly reduces in vitro Tregs suppressive function. Natural killer cells (NKs) expressed PD-1 and engagement of PD-1 reduces their cytolytic potential. To identify biomarkers informative and predictive of Nivolumab efficacy Tregs and NKs phenotype and function was determined in Nivolumab treated metastatic renal cancer (mRCC) patients (REV). Effect of CXCR4 antagonism are evaluated on ex vivo Tregs and NKs. Methods: 48 REV, 26 other than ICI treated-mRCC (CTR) and 23 Healthy Donors (HD) were enrolled. To date, the clinical evaluation is available for 42 REV [14 with objective response (OR) and 28 in progression (PD)] and 18 CTR (11 OR and 7 PD). 31 patients (pts) underwent first clinical evaluation at 3 months resulting in 25 pts with OR and 6 in PD. Tregs (CD4+CD25+CD127lowFOXP3+) and NKs (CD3-CD56+CD107a+) phenotype and function were evaluated at day 0, 14, 28, 90, and 180. CFSE-T-effector proliferation-Tregs dependent and CD107a externalization toward K562 as NKs function were evaluated. Results: Higher NKG2D was reported on CD3-CD56dim cells from OR as compared to PD pts (p=0.0054). Statistically significant low NKs basal activity was detected at T0 in 3 months PD- Nivolumab treated pts (p=0.032). CD107a+/CD53+CD56+ is higher in OR compared to PD pts over time. Percent Tregs CD4+CD25+CD127lowFOXP3+ and CD8/Tregs ratio were respectively low (p 12 months) (p Conclusions: Basal NK activity and 2 weeks Tregs evaluation discriminates mRCC Nivolumab responding patients. Ex vivo CXCR4 antagonism inhibits Tregs function in responding patients at early treatment time point (2 weeks). These evidence are not detected in non-immune treated mRCC patients. Citation Format: Sara Santagata, Anna Maria Trotta, Giuseppina Rea, Maria Napolitano, Anna Capiluongo, Crescenzo D'Alterio, Marilena Di Napoli, Sabrina Rossetti, Sandro Pignata, Stefania Scala. Basal NK activity and early Tregs inhibition predicts nivolumab responsiveness in metastatic renal cancer patients (REVOLUTION) trial [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6675.

Retrograde signaling is an intracellular communication process defined by cues generated in chloroplast and mitochondria which traverse membranes to their destination in the nucleus in order to regulate nuclear gene expression and protein synthesis. The coding and decoding of such organellar message(s) involve gene medleys and metabolic components about which more is known in higher plants than the unicellular organisms such as algae. Chlamydomonas reinhardtii is an oxygenic microalgal model for genetic and physiological studies. It harbors a single chloroplast and is amenable for generating mutants. The focus of this review is on studies that delineate retrograde signaling in Chlamydomonas vis a vis higher plants. Thus, communication networks between chloroplast and nucleus involving photosynthesis- and ROS-generated signals, functional tetrapyrrole biosynthesis intermediates, and Ca2+-signaling that modulate nuclear gene expression in this alga are discussed. Conceptually, different signaling components converge to regulate either the same or functionally-overlapping gene products.

Il presente documento ha lo scopo di illustrare le varie attività svolte nell'ambito del progetto PHOTOTECH che ha portato alla costituzione di un'ampia rete scientifica evidenziando per ciascuna attività il numero di partecipanti, i paesi partecipanti, la distribuzione dei budget per attività e per paese, per poi concludere con una analisi dei punti di forza e di debolezza nella gestione del progetto.

The integration of carbon nanotubes (CNTs) with plant machinery paved the way for various solutions to enhance plants vigor and their production efficiency [1,2]. Among others, the coupling of plant photosynthetic structures with nanomaterials has suggested the capability of single-walled CNTs (SWCNTs) to increase the efficiency of energy harnessing in the photosynthetic process [3]. This new knowledge may foster the exploitation of the nanotechnology tools to empower photosynthetic performance and production yields of commercially important microalgal species [4]. The research identified the physicochemical properties of SWCNTs enabling their entry into walled-cells of microalgae and developed experimental approaches to track their cell internalization. We tested the potential of the SWCNTs to improve algal fitness and photosynthetic performance by analysing the interaction of the carbon nanotubes and photochemical reactions in the model microorganism Chlamydomonas reinhardtii. The algal cultures were fed with stably dispersed SWCNTs and the effects of SWCNT-exposure on the function of Photosystem II and Photosystem I were studied under standard or stress-associated conditions. [1] Wang et al. 2016. Trends in Plant Science 21: 699-712. [2] Guatimosim et al. 2016. In: Bioengineering Applications of Carbon Nanostructures, A. Jorio (ed.), Springer Intern. Publ. Switzerland, pp. 17-29. [3] Giraldo et al. 2014. Nature Materials 13: 400-408. [4] Lambreva et al. 2015. Photosynthesis Research 125: 451-471.

Cationic nucleopeptides belong to a family of synthetic oligomers composed by amino acids and nucleobases. Their capability to recognize nucleic acid targets and to cross cellular membranes provided the basis for considering them as novel non-covalent delivery agents for nucleic acid pharmaceuticals. Herein, starting from a 12-mer nucleopeptide model, the number of cationic residues was modulated in order to obtain new nucleopeptides endowed with high solubility in acqueous medium, acceptable bio-stability, low cytotoxicity and good capability to bind nucleic acid. Two candidates were selected to further investigate their potential as nucleic acid carriers, showing higher efficiency to deliver PNA in comparison with RNA. Noteworthy, this study encourages the development of nucleopeptides as new carriers to extend the known strategies for those nucleic acid analogues, especially PNA, that still remain difficult to drive into the cells.

Photosystem II (PSII) reaction centre D1 protein of oxygenic phototrophs is pivotal for sustaining photosynthesis. Also, it is targeted by herbicides and herbicide-resistant weeds harbour single amino acid substitutions in D1. Conservation of D1 primary structure is seminal in the photosynthetic performance in many diverse species. In this study, we analysed built-in and environmentally-induced (high temperature and high photon fluency – HT/HL) phenotypes of two D1 mutants of Chlamydomonas reinhardtii with Ala250Arg (A250R) and Ser264Lys (S264K) substitutions. Both mutations differentially affected efficiency of electron transport and oxygen production. In addition, targeted metabolomics revealed that the mutants undergo specific differences in primary and secondary metabolism, namely, amino acids, organic acids, pigments, NAD, xanthophylls and carotenes. Levels of lutein, β-carotene and zeaxanthin were in sync with their corresponding gene transcripts in response to HT/HL stress treatment in the parental (IL) and A250R strains. D1 structure analysis indicated that, among other effects, remodelling of H-bond network at the QB site might underpin the observed phenotypes. Thus, the D1 protein, in addition to being pivotal for efficient photosynthesis, may have a moonlighting role in rewiring of specific metabolic pathways, possibly involving retrograde signalling.

The mineralization process is crucial to the load-bearing characteristics of the bone extracellular matrix. In this work, we have studied the spatiotemporal dynamics of mineral deposition by human bone marrow mesenchymal stem cells differentiating toward osteoblasts promoted by the presence of exogenous hydroxyapatite nanoparticles. At the molecular level, the added nanoparticles positively modulated the expression of bone-specific markers and enhanced calcified matrix deposition during osteogenic differentiation. The nucleation, growth and spatial arrangement of newly deposited hydroxyapatite nanocrystals have been evaluated using scanning micro X-ray diffraction and scanning micro X-ray fluorescence. As leading results, we have found the emergence of a complex scenario where the spatial organization and temporal evolution of the process exhibit heterogeneous and self-organizing dynamics. At the same time the possibility of controlling the differentiation kinetics, through the addition of synthetic nanoparticles, paves the way to empower the generation of more structured bone scaffolds in tissue engineering and to design new drugs in regenerative medicine.

Application of carbon nanotubes (CNTs) in plant biotechnology and agriculture brought to light an increasing amount of new findings, revealing the potential of CNTs to promote plant growth and crop production, or to control the delivery of fertilizers or pesticides to crops [1,2]. The coupling of plant cell structures with nanomaterials, used in the novel plant nanobionics approach, suggested the capability of CNTs to increase the efficiency of energy harnessing in the photosynthetic process and to improve cell response to oxidative stress conditions [3]. This new knowledge may foster the exploitation of the nanotechnology tools to empower photosynthetic performance and production yields of commercially important microalgal species. Large-scale cultivation of microalgae in photo-bioreactors plays an important role in the production of biomass, biofuels or high-value compounds [3]. One of the main problems in high-density-cultured algal bioreactors is the reduction of production yield due to occurrence of strong light-shading. Besides, very often, the introduction of different stress conditions (e.g. nutrients starvation) is exploited to redirect the algal metabolism towards the synthesis of desired compounds. Thus, experimental strategies taking advantages of CNT ability to assist the photosynthetic electron transport and the cell uptake of engineered nanoparticles or molecules with antioxidant or signaling functions bare great capacity to improve fitness and photosynthetic performance of commercially important photosynthetic microorganisms under large-scale manufacture conditions [4]. Here we will discuss the potential of the CNTs to enhance functions of algae facilitating a more efficient use of photosynthetic algal systems in the sustainable production of valuable goods. The research is focused on the development of experimental approaches to enable the interactions of photosynthetic microorganisms and CNTs, and to study the effects of CNT properties on algal fitness and photosynthetic performance, and nanotubes uptake into algal cell. [1] Wang et al., Trends Plant Sci. 21 (2016) 699-712. [2] Guatimosim et al., In: Bioengineering Applications of Carbon Nanostructures, A. Jorio (ed.), Springer Intern. Publ. Switzerland 2016, pp. 17-29. [3] Giraldo et al., Nature Mater. 13 (2014) 400-408. [4] Wong et al., Nature Mater. 16 (2017) 264-272. [5] Lambreva et al., Photosynth. Res. 125 (2015) 451-471.

An optical/amperometric biosensor based on algal cells immobilized in calcium alginate gel was developed. Various Chlamydomonas reinhardtii strains mutated at the level of photosynthetic D1 protein were used as biomediators to quantify the capacity of the carotenoid xanthophylls to protect the photosynthetic apparatus from photoinhibition. The highly sensitive/selective biosensor was used for studies on cell physiology aimed to determine the antioxidant and light filtering effects of the xanthophyll astaxanthin. The biosensor was proved to be suitable for the determination of the exogenous supplied astaxanthin, showing in a short time a reliable response with a detection limit of 3 μM. This technique revealed the photoprotective effect of astaxanthin-enriched extracts of Haematococcus pluvialis in algal cells. The results suggest that in algae astaxanthin exploits both filtering and antioxidant effects at different light intensities. This bioinspired approach can provide new insights into biological, biomedical, environmental and agricultural research applications and nutraceutical studies.