Integrating system biology and intratumor gene therapy by trans-complementing the appropriate co-stimulatory molecule as payload in oncolytic herpes virus.

Systems biology has been applied at the multi-scale level within the cancer field, improving cancer prevention, diagnosis and enabling precision medicine approaches. While systems biology can expand the knowledge and skills for oncological treatment, it also represents a challenging expedition due to cancer complexity, heterogeneity and diversity not only between different cancer indications, but also in its evolution process through space and time. Here, by characterizing the transcriptional perturbations of the tumor microenvironment induced by oncolytic, we aimed to rationally design a novel armed oncolytic herpes virus. We found that intratumor oncovirotherapy with HSV-1 induces T-cell activation signatures and transcriptionally activates several costimulatory molecules. We identified differentially expressed costimulatory receptors and binding partners, where inducible co-stimulators (ICOS) resulted in the potentially most beneficial targeted therapy. Through an ex-vivo transcriptomic analysis, we explored the potential of arming an oncolytic virus as a combination therapy strategy; in particular, we engineered a targeted herpes virus encoding ICOSL (THV_ICOSL), which resulted in a significant improvement in tumor size control compared to unarmed parental virus. Also, combination with a PD-1 inhibitor enhanced antitumor efficacy as predictable by upregulation of PD-1 and ligands pair (PD-L1/PD-L2) upon oncolytic virus injection. Generation of the human version of this virus encoding hICOSL orthologue effectively and specifically activated human T cells by triggering the ICOS pathway. Our data support the data-driven generation of armed oncolytic viruses as combination immunotherapeutic with checkpoint inhibitors.

Environmental risk assessment of the anionic surfactant sodium lauryl ether sulphate in site-specific conditions arising from mechanized tunnelling.

Sodium lauryl ether sulphate (SLES) is the anionic surfactant commonly utilized as the main synthetic chemical component in most foaming agents used in mechanized tunnelling. This produces huge amounts of soil debris which can contain residual concentrations of SLES. The absence of environmental quality standards for soil and water and the limited information about SLES persistence in real excavated soils do not facilitate any re-use of soil debris as by-products. The environmental risk assessment (ERA) of foaming agents containing SLES can be a valid tool for this purpose. In this study, an ERA analysis of SLES in 12 commercial formulations (cf) used for tunnelling excavation was performed. Various soils from different tunnel excavation sites were conditioned with the selected foaming agents containing SLES. Predicted or measured environmental concentrations (PECs, MECs) were determined and then compared with the Predicted No Effect Concentrations (PNECs) for both the terrestrial and aquatic compartments. The results indicate a reduction of the potential risk over time for these ecosystems, with differences depending on both the commercial foaming formulation and the spoil material characteristics. However, because potential threats to the natural environment cannot be excluded, some risk management and mitigation actions are discussed.

Spatial-temporal analysis and risk characterisation of pesticides in Alpine glacial streams.

We analysed the spatial and temporal distribution of a selection of pesticides in Alpine glaciers used on the Po Plain in Northern Italy, near the Alps. By analysing a 102-m ice core taken from the Lys Glacier (Monte Rosa massif, Italy), we highlight historical contamination from the insecticide chlorpyrifos and the herbicide terbuthylazine, confirming the role of alpine glaciers as temporal sinks. In addition, we collected meltwater samples from six glaciers distributed along the Alpine Arc during the summer of 2016, which showed widespread contamination by pesticides. Overall, chlorpyrifos and terbuthylazine dominated the contaminant fingerprint of all of the studied glaciers, with contamination peaks occurring at the beginning of the melting season. This highlights the importance of the medium-range atmospheric transport of these pesticides in connection with agricultural practices in the areas beneath the Italian Alps, where they are widely applied. The release of pesticides in meltwater can lead to potential risks to the aquatic ecosystems of headwater streams, as we demonstrate for chlorpyrifos. This suggests that the medium-range atmospheric transport of pesticides should be considered as part of regulations to protect the water quality of these pristine environments.

Optical signature of erythrocytes by light scattering in microfluidic flows.

A camera-based light scattering approach coupled with a viscoelasticity-induced cell migration technique has been used to characterize the morphological properties of erythrocytes in microfluidic flows. We have obtained the light scattering profiles (LSPs) of individual living cells in microfluidic flows over a wide angular range and matched them with scattering simulations to characterize their morphological properties. The viscoelasticity-induced 3D cell alignment in microfluidic flows has been investigated by bright-field and holographic microscopy tracking, where the latter technique has been used to obtain precise cell alignment profiles in-flow. Such information allows variable cell probability control in microfluidic flows at very low viscoelastic polymer concentrations, obtaining cell measurements that are almost physiological. Our results confirm the possibility of precise, label-free analysis of individual living erythrocytes in microfluidic flows.

Self-propelling bacteria mimic coherent light decorrelation.

We show here that live e-coli bacterial culture, thanks to the self-propelling feature, can significantly reduce the coherent noise. In fact, the typical self-propelled drive of such microorganisms provides enough time diversity in speckle patterns. Optical properties of a bacteria suspension have been investigated and analyzed thus showing that it behaves as a quite good optical speckle decorrelation device. Samples with different bacteria densities have been studied. The decorrelation effect has been demonstrated by probing the imaging performance in through transmission in coherent microscope configuration.

Imaging through scattering microfluidic channels by digital holography for information recovery in lab on chip.

We tackle the problem of information recovery and imaging through scattering microfluidic chips by means of digital holography (DH). In many cases the chip can become opalescent due to residual deposits settling down the inner channel faces, biofilm formation, scattering particle uptake by the channel cladding or its damaging by corrosive substances, or even by condensing effect on the exterior channels walls. In these cases white-light imaging is severely degraded and no information is obtainable at all about the flowing samples. Here we investigate the problem of counting and estimating velocity of cells flowing inside a scattering chip. Moreover we propose and test a method based on the recording of multiple digital holograms to retrieve improved phase-contrast images despite the strong scattering effect. This method helps, thanks to DH, to recover information which, otherwise, would be completely lost.

Random resampling masks: a non-Bayesian one-shot strategy for noise reduction in digital holography.

Holographic imaging may become severely degraded by a mixture of speckle and incoherent additive noise. Bayesian approaches reduce the incoherent noise, but prior information is needed on the noise statistics. With no prior knowledge, one-shot reduction of noise is a highly desirable goal, as the recording process is simplified and made faster. Indeed, neither multiple acquisitions nor a complex setup are needed. So far, this result has been achieved at the cost of a deterministic resolution loss. Here we propose a fast non-Bayesian denoising method that avoids this trade-off by means of a numerical synthesis of a moving diffuser. In this way, only one single hologram is required as multiple uncorrelated reconstructions are provided by random complementary resampling masks. Experiments show a significant incoherent noise reduction, close to the theoretical improvement bound, resulting in image-contrast improvement. At the same time, we preserve the resolution of the unprocessed image.

Imaging live humans through smoke and flames using far-infrared digital holography.

The ability to see behind flames is a key challenge for the industrial field and particularly for the safety field. Development of new technologies to detect live people through smoke and flames in fire scenes is an extremely desirable goal since it can save human lives. The latest technologies, including equipment adopted by fire departments, use infrared bolometers for infrared digital cameras that allow users to see through smoke. However, such detectors are blinded by flame-emitted radiation. Here we show a completely different approach that makes use of lensless digital holography technology in the infrared range for successful imaging through smoke and flames. Notably, we demonstrate that digital holography with a cw laser allows the recording of dynamic human-size targets. In this work, easy detection of live, moving people is achieved through both smoke and flames, thus demonstrating the capability of digital holography at 10.6 µm.

Multi-wavelengths digital holography: reconstruction, synthesis and display of holograms using adaptive transformation.

A method based on spatial transformations of multiwavelength digital holograms and the correlation matching of their numerical reconstructions is proposed, with the aim to improve superimposition of different color reconstructed images. This method is based on an adaptive affine transform of the hologram that permits management of the physical parameters of numerical reconstruction. In addition, we present a procedure to synthesize a single digital hologram in which three different colors are multiplexed. The optical reconstruction of the synthetic hologram by a spatial light modulator at one wavelength allows us to display all color features of the object, avoiding loss of details.

An automatic method for assembling a large synthetic aperture digital hologram.

A major issue so far for digital holography is the low spatial resolution generally achieved. The numerical aperture is limited by the area of currently available detectors, such as CCD sensors, which is significantly lower than that of a holographic plate. This is an even more severe constraint when IR sensors such as microbolometers are taken into account. In order to increase the numerical aperture of such systems, we developed an automatic technique which is capable of recording several holograms and of stitching them together, obtaining a digital hologram with a synthetic but larger numerical aperture. In this way we show that more detail can be resolved and a wider parallax angle can be achieved. The method is demonstrated for visible as well IR digital holography, recording and displaying large size objects.

Driving and analysis of micro-objects by digital holographic microscope in microfluidics.

We propose an optical configuration in which floating particles in a microfluidic chamber can be characterized by an interference microscopy configuration to obtain quantitative phase-contrast maps. The configuration is simply made by two laser beams from the same laser source. One beam provides the optical forces for driving the particle along appropriate paths, but at same time works as the object illumination beam in the holographic microscope. The second beam plays the role of the reference beam, allowing recording of an interference fringe pattern (i.e., the digital hologram) in an out-of-focus image plane. The system and method are illustrated and experimental results are offered for polymeric particles as well as for in vitro cells with the aim to demonstrate the approach.

Automatic focusing in digital holography and its application to stretched holograms.

The searching and recovering of the correct reconstruction distance in digital holography (DH) can be a cumbersome and subjective procedure. Here we report on an algorithm for automatically estimating the in-focus image and recovering the correct reconstruction distance for speckle holograms. We have tested the approach in determining the reconstruction distances of stretched digital holograms. Stretching a hologram with a variable elongation parameter makes it possible to change the in-focus distance of the reconstructed image. In this way, the proposed algorithm can be verified at different distances by dispensing the recording of different holograms. Experimental results are shown with the aim of demonstrating the usefulness of the proposed method, and a comparative analysis has been performed with respect to other existing algorithms developed for DH.

Soluble parasite antigens from Babesia canis do not directly activate the kallikrein system in dogs infected with Babesia canis.

Soluble parasite antigens (SPA) from Babesia canis have been shown to induce protective immunity when used as vaccine. In order to explain the immune mechanisms of vaccination, the precise role of SPA in the pathogenesis of canine babesiosis is under investigation. Earlier studies suggested that the plasma kallikrein system is central in the pathogenesis of babesiosis, malaria and trypanosomosis, and significant plasma kallikrein activation during acute B. bovis and P. knowlesi infections has been described. In the studies presented here dogs were experimentally infected with B. canis to investigate whether the plasma kallikrein system is activated during babesiosis infection. Results showed that prekallikrein levels decreased during episodes of peak parasitaemia. No effect was found on the kallikrein levels. In order to determine whether B. canis SPA could activate plasma kallikrein, dogs were infused with variable amounts of B. canis SPA and plasma samples were taken for (pre-) kallikrein determination. The results indicated that B. canis SPA did not affect plasma (pre-) kallikrein levels. In addition, the effect of B. canis SPA on (pre-) kallikrein levels in normal dog plasma was determined in vitro. Again, no effect on (pre-) kallikrein levels was found. The results suggest that, although the kallikrein pathway may be involved in B. canis-associated pathology, the system is not directly activated by B. canis SPA. Furthermore, infusion of B. canis SPA as well as stroma of normal dog erythrocytes triggered the production of the acute phase reactant, C-reactive protein. This suggests that the inflammatory response that is triggered during B. canis infection could be in part due to the release and exposure of self molecules. The implications of these findings are discussed.

Digital self-referencing quantitative phase microscopy by wavefront folding in holographic image reconstruction.

A completely numerical method, named digital self-referencing holography, is described to easily accomplish a quantitative phase microscopy for microfluidic devices by a digital holographic microscope. The approach works through an appropriate numerical manipulation of the retrieved complex wavefront. The self-referencing is obtained by folding the retrieved wavefront in the image plane. The folding operation allows us to obtain the correct phase map by subtracting from the complex region of interest a flat area outside the microfluidic channel. To demonstrate the effectiveness of the method, quantitative phase maps of bovine spermatozoa and in vitro cells are retrieved.

Assessing the environmental risks associated with contaminated sites: Definition of an Ecotoxicological Classification index for landfill areas (ECRIS).

Assessing ecological risk in quantitative terms is a site-specific complex procedure requiring evaluation of all possible pathways taken by the chemicals from the contamination source to the targets to be protected. Unfortunately, too many cases lack of physico-chemical and ecotoxicological data makes impossible to quantify the ecological risk. We present the Ecotoxicological Classification Risk Index for Soil (ECRIS), a new classification system specific for soil risk assessment, which gives a comparative indication of the risk linked to environmental contamination by any chemical. The tool we propose is based on the integration of a data set characterizing the ecotoxicological and exposure profile of chemicals. ECRIS is a simple approach specifically set up for the landfill scenario. This index draws on the huge amount of data from our many years of leachate analysis. ECRIS is useful for a first screening of probably contaminated soil. A case study based on some Italian landfills is proposed.

Controlling depth of focus in 3D image reconstructions by flexible and adaptive deformation of digital holograms.

We show here that through an adaptive deformation of digital holograms it is possible to manage the depth of focus in 3D imaging reconstruction. Deformation is applied to the original hologram with the aim to put simultaneously in focus, and in one reconstructed image plane, different objects lying at different distances from the hologram plane (i.e., CCD sensor). In the same way, by adapting the deformation it is possible to extend the depth of field having a tilted object entirely in focus. We demonstrate the method in both lensless as well as in microscope configuration.

Investigation of angular multiplexing and de-multiplexing of digital holograms recorded in microscope configuration.

We investigated a method for the angular multiplexing and de-multiplexing of digital holograms recorded in microscope off-axis configuration. The multiplexing has been performed rotating numerically one hologram at different angles and adding all the rotated holograms to obtain a single synthetic digital hologram. Then the digital holograms were de-multiplexed thanks to the unique property of the digital holography to manage numerically the complex wavefields at different image planes. We show that it is possible to retrieve correctly quantitative information about the amplitude and phase maps. The obtained results can be useful to employ the multiplexing technique during the recording process by rotating the CCD array.

Systemic inflammatory responses in dogs experimentally infected with Babesia canis; a haematological study.

A detailed haematological study of dogs that were infected with low, moderate or high numbers of Babesia canis-infected red blood cells was performed in an attempt to elucidate the pathogenesis early after B. canis infection. Results showed that upon infection the C-reactive protein (CRP) level in plasma increased prior to the detection of parasites in the blood indicative of an acute phase reaction. The response was further characterised by fever, fibrinogenaemia, thrombocytopenia and leucopoenia. Thrombocytopenia was associated with increased coagulation time. Infected dogs also developed life threatening hypotension, and dogs that were infected with the highest dose of B. canis-infected red blood cells had to be treated chemotherapeutically. Hypotension was associated with a reduced packed cell volume (PCV). This reduction of PCV correlated with reduced plasma creatinin concentration, suggesting that the plasma volume was increased, affecting both the erythrocyte and creatinin concentration in the plasma. Importantly, the onset of the response but not the dynamics of the response was dependent on the infectious dose i.e. curves obtained with different doses of infected erythrocytes appeared to be shifted in time but had a similar shape. This indicates that infection triggered a preset inflammatory response.

Tunable liquid microlens arrays in electrode-less configuration and their accurate characterization by interference microscopy.

A special class of tunable liquid microlenses is presented here. The microlenses are generated by an electrowetting effect under an electrode-less configuration and they exhibit two different regimes that are named here as separated lens regime (SLR) and wave-like lens regime (WLR). The lens effect is induced by the pyroelectricity of polar dielectric crystals, as was proved in principle in a previous work by the same authors (S. Grilli et al., Opt. Express 16, 8084, 2008). Compared to that work, the improvements to the experimental set-up and procedure allow to reveal the two lens regimes which exhibit different optical properties. A digital holography technique is used to reconstruct the transmitted wavefront during focusing and a focal length variation in the millimetre range is observed. The tunability of such microlenses could be of great interest to the field of micro-optics thanks to the possibility to achieve focus tuning without moving parts and thus favouring the miniaturization of the optical systems.

Numerical multiplexing and demultiplexing of digital holographic information for remote reconstruction in amplitude and phase.

We investigate the possibility to multiplexing (Mux) and demultiplexing (de-Mux) numerically digital holograms (DHs) with the aim of optimizing their storage and/or transmission process. The DHs are multiplexed and demultiplexed thanks to the unique property of the digital holography to numerically manage the complex wavefields. We show that it is possible to retrieve correctly quantitative information about the amplitude and phase of one hundred DHs. This result can be useful to transmit efficiently, in terms of reduced amount of data, the DHs from the recording head to a remote display unit.