Microfluidic Platform for Adherent Single Cell High-Throughput Screening

This paper was presented at the 4th Micro and Nano Flows Conference (MNF2014), which was held at University College, London, UK. The conference was organised by Brunel University and supported by the Italian Union of Thermofluiddynamics, IPEM, the Process Intensification Network, the Institution of Mechanical Engineers, the Heat Transfer Society, HEXAG - the Heat Exchange Action Group, and the Energy Institute, ASME Press, LCN London Centre for Nanotechnology, UCL University College London, UCL Engineering, the International NanoScience Community, www.nanopaprika.eu.Traditionally, in vitro investigations on biology and physiology of cells rely on averaging the responses eliciting from heterogeneous cell populations, thus being unsuitable for assessing individual cell behaviors in response to external stimulations. In the last years, great interest has thus been focused on single cell analysis and screening, which represents a promising tool aiming at pursuing the direct and deterministic control over cause-effect relationships guiding cell behavior. In this regard, a high-throughput microfluidic platform for trapping and culturing adherent single cells was presented. A single cell trapping mechanism was implemented based on dynamic variation of fluidic resistances. A round-shaped culture chamber (Φ=250μm, h=25μm) was conceived presenting two connections with a main fluidic path: (i) an upper wide opening, and (ii) a bottom trapping junction which modulates the hydraulic resistance. Several layouts of the chamber were designed and computationally validated for the optimization of the single cell trapping efficacy. The optimized chamber layouts were integrated in a polydimethylsiloxane (PDMS) microfluidic platform presenting two main functionalities: (i) 288 chambers for trapping single cells, and (ii) a chaoticmixer based serial dilution generator for delivering both soluble factors and non-diffusive molecules under spatio-temporally controlled chemical patterns. The devices were experimentally validated and allowed for trapping individual U87-MG (human glioblastoma-astrocytoma epithelial-like) cells and culturing them up to 3 days

Case for diagnosis. A bluish nodule on the scalp

A 32-year-old man was seen due to a 24-month history of an asymptomatic nodular lesion in the left parietal region. Physical examination revealed a smoothly bluish elevated nodule of 1.5cm x 1cm. He reported no preceding history of trauma. Dermoscopy revealed a homogeneous blue pattern (Figure 1). The tumor was surgically excised and sent for dermatopathological study. Histological examination showed a circumscribed dermal tumor without epidermal attachmen

High-throughput microfluidic platform for adherent single cells non-viral gene delivery

The widespread use of gene therapy as a therapeutic tool relies on the development of DNA-carrying vehicles devoid of any safety concerns. In contrast to viral vectors, non-viral gene carriers show promise in this perspective, although their low transfection efficiency leads to the necessity to carry out further optimizations. In order to overcome the limitations of traditional macroscale approaches, which mainly consist of time-consuming and simplified models, a microfluidic strategy has been developed to carry out transfection studies on single cells in a high-throughput and deterministic fashion. A single cell trapping mechanism has been implemented, based on the dynamic variation of fluidic resistances. For this purpose, we designed a round-shaped culture chamber integrated with a bottom trapping junction, which modulates the hydraulic resistance. Several layouts of the chamber were designed and computationally validated for optimization of the single cell trapping efficacy. The optimized chamber layout was integrated in a polydimethylsiloxane (PDMS) microfluidic platform presenting two main functionalities: (i) 288 chambers for trapping single cells, and (ii) a serial dilution generator with chaotic mixing properties, able to deliver to the chambers both soluble factors and non-diffusive particles (i.e., polymer/DNA complexes, polyplexes) under spatio-temporally controlled chemical patterns. The devices were experimentally validated and allowed the trapping of individual human glioblastoma–astrocytoma epithelial-like cells (U87-MG) with a trapping efficacy of about 40%. The cells were cultured within the device and underwent preliminary transfection experiments using 25 kDa linear polyethylenimine (lPEI)-based polyplexes, confirming the potentiality of the proposed platform for the future high-throughput screening of gene delivery vectors and for the optimization of transfection protocols

Cutaneous mucormycosis

Cutaneous mucormycosis is an emerging fungal infection caused by opportunistic fungi of the phylum Glomeromycota. It is frequent in poorly controlled diabetic patients and individuals with immunosuppression. It is usually acquired by direct inoculation through trauma. The clinical presentation is nonspecific, but an indurated plaque that rapidly evolves to necrosis is a common finding. Diagnosis should be confirmed by demonstration of the etiological agent and new molecular diagnostic tools have recently been described. It is an invasive life-threatening disease and in order to improve survival, a prompt diagnosis and multidisciplinary management should be provided. The treatment of choice is amphotericin B, but new azoles, such as posaconazole and isavuconazole, must be considered. Keywords: Mucormycosis; Mucorales; Mucor; Zygomycosis; Rhizopu

Assessment of maize nitrogen uptake from PRISMA hyperspectral data through hybrid modelling

The spaceborne imaging spectroscopy mission PRecursore IperSpettrale della Missione Applicativa (PRISMA), launched on 22 March 2019 by the Italian Space Agency, opens new opportunities in many scientific domains, including precision farming and sustainable agriculture. This new Earth Observation (EO) data stream requires new-generation approaches for the estimation of important biophysical crop variables (BVs). In this framework, this study evaluated a hybrid approach, combining the radiative transfer model PROSAIL-PRO and several machine learning (ML) regression algorithms, for the retrieval of canopy chlorophyll content (CCC) and canopy nitrogen content (CNC) from synthetic PRISMA data. PRISMA-like data were simulated from two images acquired by the airborne sensor HyPlant, during a campaign performed in Grosseto (Italy) in 2018. CCC and CNC estimations, assessed from the best performing ML algorithms, were used to define two relations with plant nitrogen uptake (PNU). CNC proved to be slightly more correlated to PNU than CCC (R-2 = 0.82 and R-2 = 0.80, respectively). The CNC-PNU model was then applied to actual PRISMA images acquired in 2020. The results showed that the estimated PNU values are within the expected ranges, and the temporal trends are compatible with plant phenology stages

Mapping Asbestos-Cement Roofing with Hyperspectral Remote Sensing over a Large Mountain Region of the Italian Western Alps

The World Health Organization estimates that 100 thousand people in the world die every year from asbestos-related cancers and more than 300 thousand European citizens are expected to die from asbestos-related mesothelioma by 2030. Both the European and the Italian legislations have banned the manufacture, importation, processing and distribution in commerce of asbestos-containing products and have recommended action plans for the safe removal of asbestos from public and private buildings. This paper describes the quantitative mapping of asbestos-cement covers over a large mountainous region of Italian Western Alps using the Multispectral Infrared and Visible Imaging Spectrometer sensor. A very large data set made up of 61 airborne transect strips covering 3263 km2 were processed to support the identification of buildings with asbestos-cement roofing, promoted by the Valle d’Aosta Autonomous Region with the support of the Regional Environmental Protection Agency. Results showed an overall mapping accuracy of 80%, in terms of asbestos-cement surface detected. The influence of topography on the classification’s accuracy suggested that even in high relief landscapes, the spatial resolution of data is the major source of errors and the smaller asbestos-cement covers were not detected or misclassified

Chitosan-Graft-Branched Polyethylenimine Copolymers: Influence of Degree of Grafting on Transfection Behavior

BACKGROUND: Successful non-viral gene delivery currently requires compromises to achieve useful transfection levels while minimizing toxicity. Despite high molecular weight (MW) branched polyethylenimine (bPEI) is considered the gold standard polymeric transfectant, it suffers from high cytotoxicity. Inversely, its low MW counterpart is less toxic and effective in transfection. Moreover, chitosan is a highly biocompatible and biodegradable polymer but characterized by very low transfection efficiency. In this scenario, a straightforward approach widely exploited to develop effective transfectants relies on the synthesis of chitosan-graft-low MW bPEIs (Chi-g-bPEI(x)) but, despite the vast amount of work that has been done in developing promising polymeric assemblies, the possible influence of the degree of grafting on the overall behavior of copolymers for gene delivery has been largely overlooked. METHODOLOGY/PRINCIPAL FINDINGS: With the aim of providing a comprehensive evaluation of the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of copolymeric vectors, we have synthesized seven Chi-g-bPEI(x) derivatives with a variable amount of bPEI grafts (minimum: 0.6%; maximum: 8.8%). Along the Chi-g-bPEI(x) series, the higher the degree of grafting, the greater the ζ-potential and the cytotoxicity of the resulting polyplexes. Most important, in all cell lines tested the intermediate degree of grafting of 2.7% conferred low cytotoxicity and higher transfection efficiency compared to other Chi-g-bPEI(x) copolymers. We emphasize that, in transfection experiments carried out in primary articular chondrocytes, Chi-g-bPEI(2.7%) was as effective as and less cytotoxic than the gold standard 25 kDa bPEI. CONCLUSIONS/SIGNIFICANCE: This work underlines for the first time the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of Chi-g-bPEI(x) copolymers. Crucially, we have demonstrated that, along the copolymer series, the fine tuning of the degree of grafting directly affected the overall charge of polyplexes and, altogether, had a direct effect on cytotoxicity

Further insights in trichothiodistrophy: a clinical, microscopic, and ultrastructural study of 20 cases and literature review

Background: Trichothiodistrophy (TTD) is a rare autosomal recessive condition that is characterized by a specific congenital hair shaft dysplasia caused by deficiency of sulfur associated with a wide spectrum of multisystem abnormalities. In this article, we study clinical, microscopic, and ultrastructural findings of 20 patients with TTD with the aim to add further insights regarding to this rare condition. Additionally, analyses of our results are compared with those extracted from the literature in order to enhance its comprehensibility. Materials and Methods: Twenty cases of TTD were included: 7 from Mexico and 14 from Spain. Clinical, microscopic, scanning electron microscopy (SEM) studies and X-ray microanalysis (XrMa) were carried out in all of them. Genetic studies were performed in all seven Mexican cases. Patients with xeroderma pigmentosum and xeroderma pigmentosum/TTD-complex were excluded. Results: Cuticular changes and longitudinal crests of the hair shaft were demonstrated. These crests were irregular, disorganized, following the hair longest axis. Hair shaft sulfur deficiency was disposed discontinuously and intermittently rather than uniformly. This severe decrease of sulfur contents was located close to the trichoschisis areas. Only five patients did not show related disturbances. Micro-dolichocephaly was observed in five cases and represented the most frequent facial dysmorphism found. It is also remarkable that all patients with urologic malformations also combined diverse neurologic disorders. Moreover, three Mexican sisters demonstrated the coexistence of scarce pubic vellus hair, developmental delay, onychodystrophy, and maxillar/mandibullar hypoplasia. Conclusions: TTD phenotype has greatly varied from very subtle forms to severe alterations such as neurologic abnormalities, blindness, lamellar ichthyosis and gonadal malformations. Herein, a multisystem study should be performed mandatorily in patients diagnosed with TTD

Cancer Immunotherapy by Blocking Immune Checkpoints on Innate Lymphocytes.

Immune checkpoints refer to a plethora of inhibitory pathways of the immune system that play a crucial role in maintaining self-tolerance and in tuning the duration and amplitude of physiological immune responses to minimize collateral tissue damages. The breakdown of this delicate balance leads to pathological conditions, including cancer. Indeed, tumor cells can develop multiple mechanisms to escape from immune system defense, including the activation of immune checkpoint pathways. The development of monoclonal antibodies, targeting inhibitory immune checkpoints, has provided an immense breakthrough in cancer therapy. Immune checkpoint inhibitors (ICI), initially developed to reverse functional exhaustion in T cells, recently emerged as important actors in natural killer (NK)-cell-based immunotherapy. Moreover, the discovery that also helper innate lymphoid cells (ILCs) express inhibitory immune checkpoints, suggests that these molecules might be targeted on ILCs, to modulate their functions in the tumor microenvironment. Recently, other strategies to achieve immune checkpoint blockade have been developed, including miRNA exploiting systems. Herein, we provide an overview of the current knowledge on inhibitory immune checkpoints on NK cells and ILCs and we discuss how to target these innate lymphocytes by ICI in both solid tumors and hematological malignancies