A Rapid Protocol of Crude RNA/DNA Extraction for RT-qPCR Detection and Quantification of 'Candidatus Phytoplasma prunorum'

Many efforts have been made to develop a rapid and sensitive method for phytoplasma and virus detection. Taking our cue from previous works, different rapid sample preparation methods have been tested and applied to Candidatus Phytoplasma prunorum (‘Ca. P. prunorum’) detection by RT-qPCR. A duplex RT-qPCR has been optimized using the crude sap as a template to simultaneously amplify a fragment of 16S rRNA of the pathogen and 18S rRNA of the host plant. The specific plant 18S rRNA internal control allows comparison and relative quantification of samples. A comparison between DNA and RNA contribution to qPCR detection is provided, showing higher contribution of the latter. The method presented here has been validated on more than a hundred samples of apricot, plum and peach trees. Since 2013, this method has been successfully applied to monitor ‘Ca. P. prunorum’ infections in field and nursery. A triplex RT-qPCR assay has also been optimized to simultaneously detect ‘Ca. P. prunorum’ and Plum pox virus (PPV) in Prunus.     

Environmental conditions at arrival to the wintering grounds and during spring migration affect population dynamics of barn swallows Hirundo rustica breeding in Northern Italy

Several populations of long-distance migratory birds are currently suffering steep demographic declines. The identification of the causes of such declines is difficult because population changes may be driven by events occurring in distant geographical areas during different phases of the annual life-cycle of migrants. Furthermore, wintering areas and migration routes of populations of small-sized species are still largely unknown, with few exceptions. In this paper we identified the critical phases of the annual life-cycle that most influence the population dynamics of a small passerine, the Barn Swallow Hirundo rustica. We used information on temporal dynamics of a population breeding in Northern Italy, whose wintering range and timing of migration have been recently described by miniaturised tracking dataloggers. Our results indicated that primary productivity in the wintering grounds in the month when most individuals arrive from autumn migration and primary productivity in an area that is probably a stopover site during spring migration, influenced population dynamics more than habitat conditions at the breeding grounds. By using annual variation in primary productivity at the wintering grounds and stopover sites as predictors, we replicated the observed interannual population changes with great accuracy. However, the steep decline recently suffered by the population could be replicated only by including a constant annual decline in the model, suggesting that changes in primary productivity only predicted the interannual variation around the long-term trend. Our study therefore suggests the existence of critical periods during wintering and migration that may have large impact on population fluctuations of migrant birds.     

The rat ErbB2 tyrosine kinase receptor produced in plants is immunogenic in mice and confers protective immunity against ErbB2+ mammary cancer

The rat ErbB2 (rErbB2) protein is a 185‐kDa glycoprotein belonging to the epidermal growth factor‐related proteins (ErbB) of receptor tyrosine kinases. Overexpression and mutations of ErbB proteins lead to several malignancies including breast, lung, pancreatic, bladder and ovary carcinomas. ErbB2 is immunogenic and is an ideal candidate for cancer immunotherapy. We investigated the possibility of expressing the extracellular (EC) domain of rErbB2 (653 amino acids, aa) in Nicotiana benthamiana plants, testing the influence of the 23 aa transmembrane (TM) sequence on protein accumulation. Synthetic variants of the rErbB2 gene portion encoding the EC domain, optimized with a human codon usage and either linked to the full TM domain (rErbB2_TM, 676 aa), to a portion of it (rErbB2‐pTM, 662 aa), or deprived of it (rErbB2_noTM, 653 aa) were cloned in the pEAQ‐HT expression vector as 6X His tag fusions. All rErbB2 variants (72–74.5 kDa) were transiently expressed, but the TM was detrimental for rErbB2 EC accumulation. rERbB2_noTM was the most expressed protein; it was solubilized and purified with Nickel affinity resin. When crude soluble extracts expressing rErbB2_noTM were administered to BALB/c mice, specific rErbB2 immune responses were triggered. A potent antitumour activity was induced when vaccinated mice were challenged with syngeneic transplantable ErbB2⁺ mammary carcinoma cells. To our knowledge, this is the first report of expression of rErbB2 in plants and of its efficacy in inducing a protective antitumour immune response, opening interesting perspectives for further immunological testing.     

How pregnancy can affect autoimmune diseases progression?

Autoimmune disorders are characterized by tissue damage, caused by self-reactivity of different effectors mechanisms of the immune system, namely antibodies and T cells. Their occurrence may be associated with genetic and/or environmental predisposition and to some extent, have implications for fertility and obstetrics. The relationship between autoimmunity and reproduction is bidirectional. This review only addresses the impact of pregnancy on autoimmune diseases and not the influence of autoimmunity on pregnancy development. Th17/Th1-type cells are aggressive and pathogenic in many autoimmune disorders and inflammatory diseases. The immunology of pregnancy underlies the role of Th2-type cytokines to maintain the tolerance of the mother towards the fetal semi-allograft. Non-specific factors, including hormonal changes, favor a switch to Th2-type cytokine profile. In pregnancy Th2, Th17/Th2 and Treg cells accumulate in the decidua but may also be present in the mother’s circulation and can regulate autoimmune responses influencing the progression of autoimmune diseases.     

Inhibiting the urokinase‐type plasminogen activator receptor system recovers STZ‐induced diabetic nephropathy

The urokinase‐type plasminogen activator (uPA) receptor (uPAR) participates to the mechanisms causing renal damage in response to hyperglycaemia. The main function of uPAR in podocytes (as well as soluble uPAR ‐(s)uPAR‐ from circulation) is to regulate podocyte function through αvβ3 integrin/Rac‐1. We addressed the question of whether blocking the uPAR pathway with the small peptide UPARANT, which inhibits uPAR binding to the formyl peptide receptors (FPRs) can improve kidney lesions in a rat model of streptozotocin (STZ)‐induced diabetes. The concentration of systemically administered UPARANT was measured in the plasma, in kidney and liver extracts and UPARANT effects on dysregulated uPAR pathway, αvβ3 integrin/Rac‐1 activity, renal fibrosis and kidney morphology were determined. UPARANT was found to revert STZ‐induced up‐regulation of uPA levels and activity, while uPAR on podocytes and (s)uPAR were unaffected. In glomeruli, UPARANT inhibited FPR2 expression suggesting that the drug may act downstream uPAR, and recovered the increased activity of the αvβ3 integrin/Rac‐1 pathway indicating a major role of uPAR in regulating podocyte function. At the functional level, UPARANT was shown to ameliorate: (a) the standard renal parameters, (b) the vascular permeability, (c) the renal inflammation, (d) the renal fibrosis including dysregulated plasminogen‐plasmin system, extracellular matrix accumulation and glomerular fibrotic areas and (e) morphological alterations of the glomerulus including diseased filtration barrier. These results provide the first demonstration that blocking the uPAR pathway can improve diabetic kidney lesion in the STZ model, thus suggesting the uPA/uPAR system as a promising target for the development of novel uPAR‐targeting approaches.     

Computational modeling of progressive damage and rupture in fibrous biological tissues: application to aortic dissection

This study analyzes the lethal clinical condition of aortic dissections from a numerical point of view. On the basis of previous contributions by Gültekin et al. (Comput Methods Appl Mech Eng 312:542–566, 2016 and 331:23–52, 2018), we apply a holistic geometrical approach to fracture, namely the crack phase-field, which inherits the intrinsic features of gradient damage and variational fracture mechanics. The continuum framework captures anisotropy, is thermodynamically consistent and is based on finite strains. The balance of linear momentum and the crack evolution equation govern the coupled mechanical and phase-field problem. The solution scheme features the robust one-pass operator-splitting algorithm upon temporal and spatial discretizations. Based on experimental data of diseased human thoracic aortic samples, the elastic material parameters are identified followed by a sensitivity analysis of the anisotropic phase-field model. Finally, we simulate an incipient propagation of an aortic dissection within a multi-layered segment of a thoracic aorta that involves a prescribed initial tear. The finite element results demonstrate a severe damage zone around the initial tear and exhibit a rather helical crack pattern, which aligns with the fiber orientation. It is hoped that the current contribution can provide some directions for further investigations of this disease.

     

Population and Single-Cell Analysis of Human Cardiogenesis Reveals Unique LGR5 Ventricular Progenitors in Embryonic Outflow Tract

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