Small extracellular vesicles released from germinated kiwi pollen (pollensomes) present characteristics similar to mammalian exosomes and carry a plant homolog of ALIX

Introduction: In the last decade, it has been discovered that allergen-bearing extracellular nanovesicles, termed “pollensomes”, are released by pollen during germination. These extracellular vesicles (EVs) may play an important role in pollen-pistil interaction during fertilization, stabilizing the secreted bioactive molecules and allowing long-distance signaling. However, the molecular composition and the biological role of these EVs are still unclear. The present study had two main aims: (I) to clarify whether pollen germination is needed to release pollensomes, or if they can be secreted also in high humidity conditions; and (II) to investigate the molecular features of pollensomes following the most recent guidelines for EVs isolation and identification. Methods: To do so, pollensomes were isolated from hydrated and germinated kiwi (Actinidia chinensis Planch.) pollen, and characterized using imaging techniques, immunoblotting, and proteomics. Results: These analyses revealed that only germinated kiwi pollen released detectable concentrations of nanoparticles compatible with small EVs for shape and protein content. Moreover, a plant homolog of ALIX, which is a well-recognized and accepted marker of small EVs and exosomes in mammals, was found in pollensomes. Discussion: The presence of this protein, along with other proteins involved in endocytosis, is consistent with the hypothesis that pollensomes could comprehend a prominent subpopulation of plant exosome-like vesicles

Canonical and truncated transglutaminase-2 regulate mucin-1 expression and androgen independency in prostate cancer cell lines

Androgen independency is associated with poor prostate cancer (PCa) survival. Here we report that silencing of transglutaminase-2 (TG2) expression by CRISPR-Cas9 is associated with upregulation of androgen receptor (AR) transcription in PCa cell lines. Knockout of TG2 reversed the migratory potential and anchorage independency of PC3 and DU145 cells and revealed a reduced level of mucin-1 (MUC1) RNA transcript through unbiased multi-omics profiling, which was restored by selective add-back of the truncated TG2 isoform (TGM2_v2). Silencing of AR resulted into increased MUC1 in TG2KO PC3 cells showing that TG2 affects transcriptional regulation of MUC1 via repressing AR expression. Treatment of PC3 WT cell line with TG2 inhibitor ZDON led to a significant increase in AR expression and decrease in MUC1. ZDON also blocked the formation of MUC1-multimers labelled with TG amine-donor substrates in reducing conditions, revealing for the first time a role for TG2, which we show to be externalised via extracellular vesicles, in MUC1 stabilisation via calcium-dependent transamidation. A specific antibody towards TGM2_v2 revealed its restricted nuclear location compared to the canonical long form of TG2 (TGM2_v1), which is predominantly cytosolic, suggesting that this form contributes to the previously suggested TG2-mediated NF-κB activation and AR transcriptional repression. As TGM2_v2 transcription was increased in biopsies of early-stage prostate adenocarcinoma (PRAD) patients compared to subjects presenting inflammatory prostatitis, and total TG2 protein expression significantly increased in PRAD versus normal tissue, the role of TG2 and its truncated form as a prostate malignancy marker is suggested. In conclusion, this investigation has provided the first unbiased discovery of a novel pathway mediated by TG2 via MUC1, which is shown to contribute to androgen insensitivity and malignancy of PCa cells and be upregulated in PCa biopsies, with potential relevance to cancer immune evasion

Characterization of amyloid-\u3b2 deposits in bovine brains

Amyloid-\u3b2 (A\u3b2) deposits are seen in aged individuals of many mammalian species that possess the same aminoacid sequence as humans. This study describes A\u3b2 deposition in 102 clinically characterized cattle brains from animals aged 0 to 20 years. Extracellular and intracellular A\u3b2 deposition was detected with 4G8 antibody in the cortex, hippocampus, and cerebellum. X-34 staining failed to stain A\u3b2 deposits, indicating the non \u3b2-pleated nature of these deposits. Western blot analysis and surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry revealed in Tris, Triton, and formic acid fractions the presence of different A\u3b2 peptides, characterized mainly by C-terminally truncated forms. Exploration of the genetic variability of APOE, PSEN1, and PSEN2 genes involved in Alzheimer's disease pathogenesis revealed several previously unreported polymorphisms. This study demonstrates certain similarities between A\u3b2 deposition patterns exhibited in cattle brains and those in the human brain in early stages of aging. Furthermore, the identification of the same A\u3b2 peptides reported in humans, but unable to form aggregates, supports the hypothesis that cattle may be protected against amyloid plaque formation

Small extracellular vesicles released from germinated kiwi pollen (pollensomes) present characteristics similar to mammalian exosomes and carry a plant homolog of ALIX

IntroductionIn the last decade, it has been discovered that allergen-bearing extracellular nanovesicles, termed “pollensomes”, are released by pollen during germination. These extracellular vesicles (EVs) may play an important role in pollen-pistil interaction during fertilization, stabilizing the secreted bioactive molecules and allowing long-distance signaling. However, the molecular composition and the biological role of these EVs are still unclear. The present study had two main aims: (I) to clarify whether pollen germination is needed to release pollensomes, or if they can be secreted also in high humidity conditions; and (II) to investigate the molecular features of pollensomes following the most recent guidelines for EVs isolation and identification.MethodsTo do so, pollensomes were isolated from hydrated and germinated kiwi (Actinidia chinensis Planch.) pollen, and characterized using imaging techniques, immunoblotting, and proteomics.ResultsThese analyses revealed that only germinated kiwi pollen released detectable concentrations of nanoparticles compatible with small EVs for shape and protein content. Moreover, a plant homolog of ALIX, which is a well-recognized and accepted marker of small EVs and exosomes in mammals, was found in pollensomes.DiscussionThe presence of this protein, along with other proteins involved in endocytosis, is consistent with the hypothesis that pollensomes could comprehend a prominent subpopulation of plant exosome-like vesicles

Extracellular transglutaminase-2, nude or associated with astrocytic extracellular vesicles, modulates neuronal calcium homeostasis

We have uncovered a novel role for astrocytes-derived extracellular vesicles (EVs) in controlling intraneuronal Ca2+ concentration ([Ca2+]i) and identified transglutaminase-2 (TG2) as a surface-cargo of astrocytes-derived EVs. Incubation of hippocampal neurons with primed astrocyte-derived EVs have led to an increase in [Ca2+]i, unlike EVs from TG2-knockout astrocytes. Exposure of neurons or brain slices to extracellular TG2 promoted a [Ca2+]i rise, which was reversible upon TG2 removal and was dependent on Ca2+ influx through the plasma membrane. Patch-clamp and calcium imaging recordings revealed TG2-dependent neuronal membrane depolarization and activation of inward currents, due to the Na+/Ca2+-exchanger (NCX) operating in the reverse mode and indirect activation of L-type VOCCs, as indicated by VOCCs/NCX pharmacological inhibitors. A subunit of Na+/K+-ATPase was selected by comparative proteomics and identified as being functionally inhibited by extracellular TG2, implicating Na+/K+-ATPase inhibition in NCX reverse mode-switching leading to Ca2+ influx and higher basal [Ca2+]i. These data suggest that reactive astrocytes control intraneuronal [Ca2+]i through release of EVs with TG2 as responsible cargo, which could have a significant impact on synaptic activity in brain inflammation

A Method to Culture GABAergic Interneurons Derived from the Medial Ganglionic Eminence

Understanding the mechanisms guiding interneuron development is a central aspect of the current research on cortical/hippocampal interneurons, which is highly relevant to brain function and pathology. In this methodological study we have addressed the setup of protocols for the reproducible culture of dissociated cells from murine medial ganglionic eminences (MGEs), to provide a culture system for the analysis of interneurons in vitro. This study includes the detailed protocols for the preparation of the dissociated cells, and for their culture on optimal substrates for cell migration or differentiation. These cultures enriched in interneurons may allow the investigation of the migratory behavior of interneuron precursors and their differentiation in vitro, up to the formation of morphologically identifiable GABAergic synapses. Live imaging of MGE–derived cells plated on proper substrates shows that they are useful to study the migratory behavior of the precursors, as well as the behavior of growth cones during the development of neurites. Most MGE-derived precursors develop into polarized GABAergic interneurons as determined by axonal, dendritic, and GABAergic markers. We present also a comparison of cells from WT and mutant mice as a proof of principle for the use of these cultures for the analysis of the migration and differentiation of GABAergic cells with different genetic backgrounds. The culture enriched in interneurons described here represents a useful experimental system to examine in a relatively easy and fast way the morpho-functional properties of these cells under physiological or pathological conditions, providing a powerful tool to complement the studies in vivo

Sustained post-rituximab B cell depletion is common in ANCA-associated Vasculitis and affected by sex and renal function

Objective Despite the increasing use of rituximab in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), it remains unclear what the optimal dosing is, especially for maintenance of remission. A deeper understanding of post-rituximab B-cell repopulation patterns may aid better-tailored treatment.Methods This is a monocentric, retrospective study including ANCA-positive AAV patients receiving a single course of rituximab induction. CD19+ B cells were longitudinally monitored with flow cytometry. B-cell repopulation was defined as CD19+ >10 cells/mu L.Results Seventy-one patients were included, the majority with microscopic polyangiitis (75%), myeloperoxidase-ANCA positivity (75%) and with renal involvement (79%). During a median follow-up of 54 months since the first rituximab infusion, 44 patients (62%) repopulated B cells, with a median time to repopulation of 39 months (range 7-102). Patients experiencing B-cell depletion lasting longer than the overall median time to repopulation (39 months) exhibited a lower risk of flare and higher risk of serious infection. In multivariate Cox regression, higher estimated glomerular filtration rate (eGFR) [hazard ratio (HR) 1.84, 95% confidence interval (CI) 1.13-2.98 per 30 mL/min/1.73 m2 eGFR] and female sex (HR 2.70, 95% CI 1.37-5.31) were independent predictors of increased rate of B-cell repopulation.Conclusion A subset of AAV patients develop sustained post-rituximab B-cell depletion, which associates with reduced risk of flare and increased risk of serious infection in the long term. Preserved renal function and female sex are associated with faster B-cell repopulation. These observations further highlight the need to personalize immunosuppression to improve clinical outcomes

Diagnosis of human prion disease using real-time quaking-induced conversion testing of olfactory mucosa and cerebrospinal fluid samples

Importance: Early and accurate in vivo diagnosis of Creutzfeldt-Jakob disease (CJD) is necessary for quickly distinguishing treatable from untreatable rapidly progressive dementias and for future therapeutic trials. This early diagnosis is becoming possible using the real-time quaking-induced conversion (RT-QuIC) seeding assay, which detects minute amounts of the disease-specific pathologic prion protein in cerebrospinal fluid (CSF) or olfactory mucosa (OM) samples. Objective: To develop an algorithm for accurate and early diagnosis of CJD by using the RT-QuIC assay on CSF samples, OM samples, or both. Design, Setting, and Participants: In this case-control study, samples of CSF and OM were collected from 86 patients with a clinical diagnosis of probable (n\u2009=\u200951), possible (n\u2009=\u200924), or suspected (n\u2009=\u200911) CJD and 104 negative control samples (54 CSF and 50 OM). The CSF and OM samples were analyzed using conventional RT-QuIC. The CSF samples underwent further testing using improved RT-QuIC conditions. In addition, the diagnostic performance of a novel, easy-to-use, gentle flocked swab for sampling of OM was evaluated. Data were collected from January 1 to June 30, 2015. Main Outcome and Measures: Correlations between RT-QuIC results and the final diagnosis of recruited patients. Results: Among the 86 patients (37 men [43%] and 49 women [57%]; mean [SD] age, 65.7 [11.5] years) included for analysis, all 61 patients with sporadic CJD had positive RT-QuIC findings using OM or CSF samples or both for an overall RT-QuIC diagnostic sensitivity of 100% (95% CI, 93%-100%). All patients with a final diagnosis of non-prion disease (71 CSF and 67 OM samples) had negative RT-QuIC findings for 100% specificity (95% CI, 94%-100%). Of 8 symptomatic patients with various mutations causing CJD or Gerstmann-Str\ue4ussler-Scheinker syndrome, 6 had positive and 2 had negative RT-QuIC findings for a sensitivity of 75% (95% CI, 36%-96%). Conclusions and Relevance: A proposed diagnostic algorithm for sporadic CJD combines CSF and OM RT-QuIC testing to provide virtually 100% diagnostic sensitivity and specificity in the clinical phase of the disease. OM RT-QuIC testing to provide virtually 100% diagnostic sensitivity and specificity in the clinical phase of the disease