Rapid and Affordable High Throughput Screening of SARS-CoV-2 Variants Using Denaturing High-Performance Liquid Chromatography Analysis

Mutations in the receptor binding domain (RBD) of SARS-CoV-2 alter the infectivity, pathogenicity, and transmissibility of new variants of concern (VOCs). In addition, those mutations cause immune escape, undermining the population immunity induced by ongoing mass vaccination programs. There is an urgent need for novel strategies and techniques aimed at the surveillance of the active emergence and spread of the VOCs. The aim of this study was to provide a quick, cheap and straightforward denaturing high-performance liquid chromatography (DHPLC) method for the prompt identification of the SARS-CoV-2 VOCs. Two PCRs were designed to target the RBD region, spanning residues N417 through N501 of the Spike protein. Furthermore, a DHPLC screening analysis was set up. The screening consisted of mixing the unknown sample with a standard sample of a known variant, denaturing at high temperature, renaturing at room temperature followed by a 2-minute run using the WAVE DHPLC system to detect the heteroduplexes which invariably form whenever the unknown sample has a nucleotide difference with respect to the standard used. The workflow was able to readily detect all the variants including B.1.1.7, P.1, B.1.585 B.1. 617.2 and lineages at a very affordable cost. The DHPLC analysis was robust being able to identify variants, even in the case of samples with very unbalanced target concentrations including those samples at the limit of detection. This approach has the potential of greatly expediting surveillance of the SARS-CoV-2 variants

Mast Cell: An Emerging Partner in Immune Interaction

Mast cells (MCs) are currently recognized as effector cells in many settings of the immune response, including host defense, immune regulation, allergy, chronic inflammation, and autoimmune diseases. MC pleiotropic functions reflect their ability to secrete a wide spectrum of preformed or newly synthesized biologically active products with pro-inflammatory, anti-inflammatory and/or immunosuppressive properties, in response to multiple signals. Moreover, the modulation of MC effector phenotypes relies on the interaction of a wide variety of membrane molecules involved in cell–cell or cell-extracellular-matrix interaction. The delivery of co-stimulatory signals allows MC to specifically communicate with immune cells belonging to both innate and acquired immunity, as well as with non-immune tissue-specific cell types. This article reviews and discusses the evidence that MC membrane-expressed molecules play a central role in regulating MC priming and activation and in the modulation of innate and adaptive immune response not only against host injury, but also in peripheral tolerance and tumor-surveillance or -escape. The complex expression of MC surface molecules may be regarded as a measure of connectivity, with altered patterns of cell–cell interaction representing functionally distinct MC states. We will focalize our attention on roles and functions of recently discovered molecules involved in the cross-talk of MCs with other immune partners

Molecular and cellular environment that leads to the generation of IL-10\u2013producing B cells

L\u2019attivit\ue0 di ricerca del mio dottorato si \ue8 incentrata sullo studio dei linfociti B e, nello specifico, delle cellule B con funzioni regolatorie (CD19+ IL-10+). Gli scopi del lavoro sono stati la caratterizzazione del microambiente che induce il fenotipo regolatorio in cellule B di topo e lo studio del possibile ruolo del mastocita nella espansione e differenziazione dei linfociti B competenti alla produzione di IL-1

Ambidextrous organizations for sustainable development: The case of fair-trade systems

The fair-trade system is concerned with responsible production and consumption patterns, and may therefore contribute to the achievement of sustainable development. To ensure the functioning of this system, there exist intermediate organizations that act as a bridge between highly different and partially asymmetric contexts, such as manufacturing companies from emerging countries and profit companies from developed countries. Several studies have already shown the role these organizations play in promoting the creation of manufacturing companies in less developed countries and in fostering the introduction in western markets of goods deriving from this more sustainable supply chain. The literature, however, has still not dealt with the specific role of intermediate organizations from a strategic, long-term perspective. Adopting ambidexterity theory, the aim of the research is to investigate whether a fair-trade intermediate organization can be defined ambidextrous in the sense that it simultaneously pursues exploration and exploitation activities and sustainability and profitability related goals. Subsequently, the research aims also to understand the modalities in which explorative and exploitative competences are shared and transferred within the organizations belonging the same fair-trade system. To achieve these purposes, the study adopts a qualitative approach, based on in-depth interviews, besides the analysis of financial performances of seven organizations: one intermediate organization, three manufacturing companies from emerging countries and three buyer companies downstream in the supply chain. Results show that the intermediate organization encourages innovation and ensures efficiency and it engages in ambidexterity to favor in turn the reduction of the asymmetry of partner companies. The balance between exploration and exploitation inside the intermediate organization is however still to be achieved. The research adds insights to current knowledge about ambidexterity in practice and, as first time, it applies ambidexterity theory to the fair-trade system. In addition, direct managerial implications for the entire fair-trade system can be derived

Use of cocultures for the study of cellular interactions influencing B cell regulatory functions

Many immunological processes are contextually controlled by complex interactions among different cell types. Several studies have shown that B cells produce the immune regulatory cytokine IL-10 in response to different external stimuli but also to immune-mediated signals. Endogenous signals that derive from the cross talk between B lymphocytes and other cells of the immune system can affect IL-10 production by B cells in both physiological and pathological conditions. With the aim to provide a guide for the study of how partner cells can induce IL-10-producing B cells, here we describe the protocols to investigate IL-10 production at a single cell level in a dendritic cell-B cell coculture in vitro system. These methods are a proof of concept that can be easily extrapolated and adapted to the study of the interaction between B cells and other immune cell types

Integrating innate and adaptive immune cells: Mast cells as crossroads between regulatory and effector B and T cells

A diversity of immune mechanisms have evolved to protect normal tissues from infection, but from immune damage too. Innate cells, as well as adaptive cells, are critical contributors to the correct development of the immune response and of tissue homeostasis. There is a dynamic "cross-talk" between the innate and adaptive immunomodulatory mechanisms for an integrated control of immune damage as well as the development of the immune response. Mast cells have shown a great plasticity, modifying their behavior at different stages of immune response through interaction with effector and regulatory populations of adaptive immunity. Understanding the interplays among T effectors, regulatory T cells, B cells and regulatory B cells with mast cells will be critical in the future to assist in the development of therapeutic strategies to enhance and synergize physiological immune-modulator and -suppressor elements in the innate and adaptive immune system