Molecular detection of Grapevine fleck virus-like viruses

Molecular reagents have been developed for virus-specific and simultaneous (virus-non-specific) detection of Grapevine fleck virus (GFkV) and allied viruses, ie. Grapevine asteroid mosaic-associated virus (GAMaV) and Grapevine red globe virus (GRGV). Degenerate primers designed on nucleotide sequences of the RNA-dependent RNA polymerase (RD) and methyltransferase (MTR) domains of the GFkV genome, were able to give amplification products of the expected size from total nucleic acid extracts of:vines infected with GFkV, GAMaV, and GRGV;a Californian grapevine accession infected by a marafi-like virus;Greek grapevine accessions infected by an unidentified agent that induced symptoms reminiscent of those elicited by GAMaV in Vitis rupestris.Degenerate primers designed on the nucleotide sequence of the helicase (HEL) domain of the GFLV genome recognized all the above viruses except for GAMaV and the unidentified Greek viral agent. RD primer set worked well also with crude grapevine cortical scrapings, thus constituting a useful universal reagent for the non-specific molecular identification of GFkV-like viruses in Vitis . The marafi-like virus from California was amplified by all sets of primers, but was recognized only by the GRGV-specific probe, suggesting that it is a likely isolate of GRGV: Likewise, the unidentified virus from Greek vines shared sequence homology with GFkV and allied viruses (GAMaV and GRGV) but exhibited differences relevant enough that call for further investigations to establish its taxonomic position. While GRGV was identified, though with a very low incidence, in some 11 southern Italian grapevine cultivars, no evidence was obtained for infection by GAMaV in any of 50 cultivars analyzed.

Validation of four automatic devices for self-measurement of blood pressure according to the international protocol of the European Society of Hypertension

Jirar Topouchian1, Davide Agnoletti1, Jacques Blacher1, Ahmed Youssef1, Isabel Ibanez2,3, Jose Khabouth2, Salwa Khawaja2, Layale Beaino2, Roland Asmar1–31Centre de Diagnostic, Hôpital Hôtel-Dieu, Paris, France; 2Hôpital Libanais and Faculté Libanaise de Médecine, Beirut, Lebanon; 3Foundation-Medical Research Institutes, Geneva, SwitzerlandBackground: Four oscillometric devices for self-measurement of blood pressure (SBPM) were evaluated according to the European Society of Hypertension (ESH) international protocol and its 2010 revision in four separate studies. The Omron® M2, Omron M3, and Omron M6 measure blood pressure (BP) at the brachial level, while the Omron R2 measures BP at the wrist level.Methods: The international protocol requires a total number of 33 subjects in which the validation is performed. The Omron M2 and Omron R2 were validated in 2009 according to the ESH international protocol, while the Omron M3 and Omron M6 were validated in 2010–2011 according to the 2010 ESH international protocol revision. The protocol procedures were followed precisely.Results: All four tested devices passed the validation process. The mean differences between the device and mercury readings were 2.7 ± 5.0 and –1.4 ± 3.2 mmHg for systolic and diastolic BP, respectively, using the Omron M2 device, and 1.7 ± 3.2 and –0.9 ± 2.6 mmHg using the Omron M3, 1.6 ± 2.9 and -0.9 ± 2.5 mmHg using the Omron M6, and –1.1 ± 4.8 and –0.9 ± 4.3 mmHg using the Omron R2.Conclusion: Readings from the Omron M2, Omron M3, Omron M6, and Omron R2, differing by less than 5, 10, and 15 mmHg, fulfill the ESH international protocol and its 2010 revision requirements. Therefore, each of these four devices can be used by patients for SBPM.Keywords: Omron R2, M2, M3, M6, blood pressure measurement, validation, international protocol, European Society of Hypertensio

ETV4 and ETV5 drive synovial sarcoma through cell cycle and DUX4 embryonic pathway control

Synovial sarcoma is an aggressive malignancy with no effective treatments for patients with metastasis. The synovial sarcoma fusion SS18-SSX, which recruits the SWI/SNF-BAF chromatin remodeling and polycomb repressive complexes, results in epigenetic activation of FGF receptor (FGFR) signaling. In genetic FGFR-knockout models, culture, and xenograft synovial sarcoma models treated with the FGFR inhibitor BGJ398, we show that FGFR1, FGFR2, and FGFR3 were crucial for tumor growth. Transcriptome analyses of BGJ398-treated cells and histological and expression analyses of mouse and human synovial sarcoma tumors revealed prevalent expression of two ETS factors and FGFR targets, ETV4 and ETV5. We further demonstrate that ETV4 and ETV5 acted as drivers of synovial sarcoma growth, most likely through control of the cell cycle. Upon ETV4 and ETV5 knockdown, we observed a striking upregulation of DUX4 and its transcriptional targets that activate the zygotic genome and drive the atrophy program in facioscapulohumeral dystrophy patients. In addition to demonstrating the importance of inhibiting all three FGFRs, the current findings reveal potential nodes of attack for the cancer with the discovery of ETV4 and ETV5 as appropriate biomarkers and molecular targets, and activation of the embryonic DUX4 pathway as a promising approach to block synovial sarcoma tumors

Thiopalmitoylation of altered peptide ligands enhances their protective effects in an animal model of multiple sclerosis

Previously, we have shown that conjugation of a palmitic chain via a thioester bond to a cysteine residue in weakly or nonencephalitogenic or neuritogenic peptides markedly enhances their ability to induce autoimmune disease in an MHC class II–restricted manner. From those studies, however, it was not clear whether thiopalmitoylation of the peptides was merely enhancing their disease-inducing potential or whether the lipid was itself playing a pathogenic role. To investigate this further, we have now tested the effects of thiopalmitoylation on MHC class II–restricted altered peptide ligands (APLs), which are normally protective in experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis. We hypothesized that if thiopalmitoylation of a peptide merely enhances its innate potential, then thiopalmitoylated APLs (S-palmAPLs) should show enhanced protective effects. Alternatively, if thiopalmitoylation itself can make a peptide pathogenic, then S-palmAPLs should have decreased therapeutic potential. We synthesized APLs and corresponding S-palmAPLs and showed that the S-palmAPLs were much more effective than the nonconjugated APL at inhibiting the development of experimental autoimmune encephalomyelitis. This was due to several features of the S-palmAPL:S-palmAPL–primed cells show an enhanced ability to proliferate and produce the anti-inflammatory cytokine, IL-10, in vitro. Furthermore, the bioavailability of S-palmAPL was greatly enhanced, compared with the nonpalmitoylated APL, and S-palm APL was taken up more rapidly into dendritic cells and channeled into the MHC class II processing pathway. These results show that thiopalmitoylation of MHC class II–restricted peptides is a simple way to enhance their effects in vivo and could have wide therapeutic application

α-Synuclein Radiotracer Development and In Vivo Imaging: Recent Advancements and New Perspectives

α-Synucleinopathies including idiopathic Parkinson's disease, dementia with Lewy bodies and multiple systems atrophy share overlapping symptoms and pathological hallmarks. Selective neurodegeneration and Lewy pathology are the main hallmarks of α-synucleinopathies. Currently, there is no imaging biomarker suitable for a definitive early diagnosis of α-synucleinopathies. Although dopaminergic deficits detected with single-photon emission computed tomography (SPECT) and positron emission tomography (PET) radiotracers can support clinical diagnosis by confirming the presence of dopaminergic neurodegeneration, dopaminergic imaging cannot visualize the preceding disease process, nor distinguish α-synucleinopathies from tauopathies with dopaminergic neurodegeneration, especially at early symptomatic disease stage when clinical presentation is often overlapping. Aggregated α-synuclein (αSyn) could be a suitable imaging biomarker in α-synucleinopathies, because αSyn aggregation and therefore, Lewy pathology is evidently an early driver of α-synucleinopathies pathogenesis. Additionally, several antibodies and small molecule compounds targeting aggregated αSyn are in development for therapy. However, there is no way to directly measure if or how much they lower the levels of aggregated αSyn in the brain. There is clearly a paramount diagnostic and therapeutic unmet medical need. To date, aggregated αSyn and Lewy pathology inclusion bodies cannot be assessed ante-mortem with SPECT or PET imaging because of the suboptimal binding characteristics and/or physicochemical properties of current radiotracers. The aim of this narrative review is to highlight the suitability of aggregated αSyn as an imaging biomarker in α-synucleinopathies, the current limitations with and lessons learned from αSyn radiotracer development, and finally to propose antibody-based ligands for imaging αSyn aggregates as a complementary tool rather than an alternative to small molecule ligands. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson Movement Disorder Society.</p

Pretargeted PET Imaging with a TCO-Conjugated Anti-CD44v6 Chimeric mAb U36 and [Zr-89]Zr-DFO-PEG(5)-Tz

The recent advances in the production of engineered antibodies have facilitated the development and application of tailored, target-specific antibodies. Positron emission tomography (PET) of these antibody-based drug candidates can help to better understand their in vivo behavior. In this study, we report an in vivo proof-ofconcept pretargeted immuno-PET study where we compare a pretargeting vs targeted approach using a new Zr-89-labeled tetrazine as a bio-orthogonal ligand in an inverse electron demand Diels-Alder (IEDDA) in vivo click reaction. A CD44v6-selective chimeric monoclonal U36 was selected as the targeting antibody because it has potential in immuno-PET imaging of head-and-neck squamous cell carcinoma (HNSCC). Zirconium-89 (t(1/2) = 78.41 h) was selected as the radionuclide of choice to be able to make a head-to-head comparison of the pretargeted and targeted approaches. [Zr-89]Zr-DFO-PEG S -Tz ([Zr-89]Zr-3) was synthesized and used in pretargeted PET imaging of HNSCC xenografts (VU-SCC-OE) at 24 and 48 h after administration of a trans-cyclooctene (TCO)-functionalized U36. The pretargeted approach resulted in lower absolute tumor uptake than the targeted approach (1.5 +/- 0.2 vs 17.1 +/- 3.0% ID/g at 72 h p.i. U36) but with comparable tumor-to-non-target tissue ratios and significantly lower absorbed doses. In conclusion, anti-CD44v6 monoclonal antibody U36 was successfully used for Zr-89-immuno-PET imaging of HNSCC xenograft tumors using both a targeted and pretargeted approach. The results not only support the utility of the pretargeted approach in immuno-PET imaging but also demonstrate the challenges in achieving optimal in vivo IEDDA reaction efficiencies in relation to antibody pharmacokinetics.Peer reviewe

The P2X(7) receptor tracer [C-11]SMW139 as an in vivo marker of neuroinflammation in multiple sclerosis: a first-in man study

Purpose: The novel PET tracer [11C]SMW139 binds with high affinity to the P2X7 receptor, which is expressed on pro-inflammatory microglia. The purposes of this first in-man study were to characterise pharmacokinetics of [11C]SMW139 in patients with active relapsing remitting multiple sclerosis (RRMS) and healthy controls (HC) and to evaluate its potential to identify in vivo neuroinflammation in RRMS. / Methods: Five RRMS patients and 5 age-matched HC underwent 90-min dynamic [11C]SMW139 PET scans, with online continuous and manual arterial sampling to generate a metabolite-corrected arterial plasma input function. Tissue time activity curves were fitted to single- and two-tissue compartment models, and the model that provided the best fits was determined using the Akaike information criterion. / Results: The optimal model for describing [11C]SMW139 kinetics in both RRMS and HC was a reversible two-tissue compartment model with blood volume parameter and with the dissociation rate k4 fixed to the whole-brain value. Exploratory group level comparisons demonstrated an increased volume of distribution (VT) and binding potential (BPND) in RRMS compared with HC in normal appearing brain regions. BPND in MS lesions was decreased compared with non-lesional white matter, and a further decrease was observed in gadolinium-enhancing lesions. In contrast, increased VT was observed in enhancing lesions, possibly resulting from disruption of the blood-brain barrier in active MS lesions. In addition, there was a high correlation between parameters obtained from 60- to 90-min datasets, although analyses using 60-min data led to a slight underestimation in regional VT and BPND values. / Conclusions: This first in-man study demonstrated that uptake of [11C]SMW139 can be quantified with PET using BPND as a measure for specific binding in healthy controls and RRMS patients. Additional studies are warranted for further clinical evaluation of this novel neuroinflammation tracer

Pretargeted PET Imaging with a TCO-Conjugated Anti-CD44v6 Chimeric mAb U36 and [Zr-89]Zr-DFO-PEG(5)-Tz

The recent advances in the production of engineered antibodies have facilitated the development and application of tailored, target-specific antibodies. Positron emission tomography (PET) of these antibody-based drug candidates can help to better understand their in vivo behavior. In this study, we report an in vivo proof-ofconcept pretargeted immuno-PET study where we compare a pretargeting vs targeted approach using a new Zr-89-labeled tetrazine as a bio-orthogonal ligand in an inverse electron demand Diels-Alder (IEDDA) in vivo click reaction. A CD44v6-selective chimeric monoclonal U36 was selected as the targeting antibody because it has potential in immuno-PET imaging of head-and-neck squamous cell carcinoma (HNSCC). Zirconium-89 (t(1/2) = 78.41 h) was selected as the radionuclide of choice to be able to make a head-to-head comparison of the pretargeted and targeted approaches. [Zr-89]Zr-DFO-PEG S -Tz ([Zr-89]Zr-3) was synthesized and used in pretargeted PET imaging of HNSCC xenografts (VU-SCC-OE) at 24 and 48 h after administration of a trans-cyclooctene (TCO)-functionalized U36. The pretargeted approach resulted in lower absolute tumor uptake than the targeted approach (1.5 +/- 0.2 vs 17.1 +/- 3.0% ID/g at 72 h p.i. U36) but with comparable tumor-to-non-target tissue ratios and significantly lower absorbed doses. In conclusion, anti-CD44v6 monoclonal antibody U36 was successfully used for Zr-89-immuno-PET imaging of HNSCC xenograft tumors using both a targeted and pretargeted approach. The results not only support the utility of the pretargeted approach in immuno-PET imaging but also demonstrate the challenges in achieving optimal in vivo IEDDA reaction efficiencies in relation to antibody pharmacokinetics

Preterm Delivery Disrupts the Developmental Program of the Cerebellum

A rapid growth in human cerebellar development occurs in the third trimester, which is impeded by preterm delivery. The goal of this study was to characterize the impact of preterm delivery on the developmental program of the human cerebellum. Still born infants, which meant that all development up to that age had taken place in-utero, were age paired with preterm delivery infants, who had survived in an ex-utero environment, which meant that their development had also taken place outside the uterus. The two groups were assessed on quantitative measures that included molecular markers of granule neuron, purkinje neuron and bergmann glia differentiation, as well as the expression of the sonic hedgehog signaling pathway, that is important for cerebellar growth. We report that premature birth and development in an ex-utero environment leads to a significant decrease in the thickness and an increase in the packing density of the cells within the external granular layer and the inner granular layer well, as a reduction in the density of bergmann glial fibres. In addition, this also leads to a reduced expression of sonic hedgehog in the purkinje layer. We conclude that the developmental program of the cerebellum is specifically modified by events that follow preterm delivery