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.     

The Computational Development of Reinforcement Learning during Adolescence

Adolescence is a period of life characterised by changes in learning and decision-making. Learning and decision-making do not rely on a unitary system, but instead require the coordination of different cognitive processes that can be mathematically formalised as dissociable computational modules. Here, we aimed to trace the developmental time-course of the computational modules responsible for learning from reward or punishment, and learning from counterfactual feedback. Adolescents and adults carried out a novel reinforcement learning paradigm in which participants learned the association between cues and probabilistic outcomes, where the outcomes differed in valence (reward versus punishment) and feedback was either partial or complete (either the outcome of the chosen option only, or the outcomes of both the chosen and unchosen option, were displayed). Computational strategies changed during development: whereas adolescents’ behaviour was better explained by a basic reinforcement learning algorithm, adults’ behaviour integrated increasingly complex computational features, namely a counterfactual learning module (enabling enhanced performance in the presence of complete feedback) and a value contextualisation module (enabling symmetrical reward and punishment learning). Unlike adults, adolescent performance did not benefit from counterfactual (complete) feedback. In addition, while adults learned symmetrically from both reward and punishment, adolescents learned from reward but were less likely to learn from punishment. This tendency to rely on rewards and not to consider alternative consequences of actions might contribute to our understanding of decision-making in adolescence.     

Phosphorylation of the amino-terminus of the AGC kinase Gad8 prevents its interaction with TORC2

Cell proliferation, metabolism, migration and survival are coordinated through the tight control of two target of rapamycin (TOR) kinase complexes: TORC1 and TORC2. Here, we show that a novel phosphorylation of fission yeast Gad8 (AGC kinase) on the evolutionarily conserved threonine 6 (Thr6) prevents the physical association between Gad8 and TORC2. Accordingly, this block to protein interactions by Gad8 Thr6 phosphorylation decreases TORC2-controlled activation of Gad8. Likewise, phosphorylation of Gad8 Thr6, possibly by PKC, prevents the association of Gad8 with TORC2 thereby increasing TORC2 activity, because it reduces Gad8-mediated feedback inhibition of TORC2. Consistently, the introduction of a Gad8 T6D mutant, that mimics phosphorylation, increased TORC2 activity. Increased PKC^Pck2 expression prevented Gad8–TORC2 binding and so reduced the TORC2-mediated phosphorylation of Gad8 serine 546 that activates Gad8. Interestingly, independent of the Ser546 phosphorylation status, Gad8 Thr6 phosphorylation is important for remodelling the actin cytoskeleton and survival upon potassium ion and heat stresses. In contrast, Ser546 phosphorylation is required for the control of G1 arrest, mating, cell length at division and vascular size. Finally, these findings reveal a novel mode of TORC2 activation that is essential for cell survival following stress.     

Pre-Osteoblasts Stimulate Migration of Breast Cancer Cells via the HGF/MET Pathway

Introduction

The occurrence of skeletal metastases in cancer, e.g. breast cancer (BC), deteriorates patient life expectancy and quality-of-life. Current treatment options against tumor-associated bone disease are limited to anti-resorptive therapies and aimed towards palliation. There remains a lack of therapeutic approaches, which reverse or even prevent the development of bone metastases. Recent studies demonstrate that not only osteoclasts (OCs), but also osteoblasts (OBs) play a central role in the pathogenesis of skeletal metastases, partly by producing hepatocyte growth factor (HGF), which promotes tumor cell migration and seeding into the bone. OBs consist of a heterogeneous cell pool with respect to their maturation stage and function. Recent studies highlight the critical role of pre-OBs in hematopoiesis. Whether the development of bone metastases can be attributed to a particular OB maturation stage is currently unknown.

Methods and Results

Pre-OBs were generated from healthy donor (HD)-derived bone marrow stromal cells (BMSC) as well as the BMSC line KM105 and defined as ALP^low OPN^low RUNX2^high OSX ^high CD166^high. Conditioned media (CM) of pre-OBs, but not of undifferentiated cells or mature OBs, enhanced migration of metastatic BC cells. Importantly, HGF mRNA was significantly up-regulated in pre-OBs versus mature OBs, and CM of pre-OBs activated the MET signaling pathway. Highlighting a key role for HGF, CM from HGF-negative pre-OBs derived from the BMSC line HS27A did not support migration of BC cells. Genetically (siMET) or pharmacologically (INCB28060) targeting MET inhibited both HGF- and pre-OB CM- mediated BC cell migration.

Conclusions

Our data demonstrate for the first time a role for pre-OBs in mediating HGF/MET- dependent migration of BC cells and strongly support the clinical evaluation of INCB28060 and other MET inhibitors to limit and/or prevent BC-associated bone metastases.     

A monoclonal antibody specific for prophase phosphorylation of histone deacetylase 1: a readout for early mitotic cells

Histone deacetylases (HDACs) are modification enzymes that regulate a plethora of biological processes. HDAC1, a crucial epigenetic modifier, is deregulated in cancer and subjected to a variety of post-translational modifications. Here, we describe the generation of a new monoclonal antibody that specifically recognizes a novel highly dynamic prophase phosphorylation of serine 406-HDAC1, providing a powerful tool for detecting early mitotic cells.     

Dynamic Modelling of Tooth Deformation Using Occlusal Kinematics and Finite Element Analysis

Background

Dental biomechanics based on finite element (FE) analysis is attracting enormous interest in dentistry, biology, anthropology and palaeontology. Nonetheless, several shortcomings in FE modeling exist, mainly due to unrealistic loading conditions. In this contribution we used kinematics information recorded in a virtual environment derived from occlusal contact detection between high resolution models of an upper and lower human first molar pair (M¹ and M₁, respectively) to run a non-linear dynamic FE crash colliding test.

Methodology

MicroCT image data of a modern human skull were segmented to reconstruct digital models of the antagonistic right M¹ and M₁ and the dental supporting structures. We used the Occlusal Fingerprint Analyser software to reconstruct the individual occlusal pathway trajectory during the power stroke of the chewing cycle, which was applied in a FE simulation to guide the M₁ 3D-path for the crash colliding test.

Results

FE analysis results showed that the stress pattern changes considerably during the power stroke, demonstrating that knowledge about chewing kinematics in conjunction with a morphologically detailed FE model is crucial for understanding tooth form and function under physiological conditions.

Conclusions/Significance

Results from such advanced dynamic approaches will be applicable to evaluate and avoid mechanical failure in prosthodontics/endodontic treatments, and to test material behavior for modern tooth restoration in dentistry. This approach will also allow us to improve our knowledge in chewing-related biomechanics for functional diagnosis and therapy, and it will help paleoanthropologists to illuminate dental adaptive processes and morphological modifications in human evolution.     

Cerebral Blood Volume ASPECTS Is the Best Predictor of Clinical Outcome in Acute Ischemic Stroke: A Retrospective,Combined Semi-Quantitative and Quantitative Assessment

Introduction

The capability of CT perfusion (CTP) Alberta Stroke Program Early CT Score (ASPECTS) to predict outcome and identify ischemia severity in acute ischemic stroke (AIS) patients is still questioned.

Methods

62 patients with AIS were imaged within 8 hours of symptom onset by non-contrast CT, CT angiography and CTP scans at admission and 24 hours. CTP ASPECTS was calculated on the affected hemisphere using cerebral blood flow (CBF), cerebral blood volume (CBV) and mean transit time (MTT) maps by subtracting 1 point for any abnormalities visually detected or measured within multiple cortical circular regions of interest according to previously established thresholds. MTT-CBV ASPECTS was considered as CTP ASPECTS mismatch. Hemorrhagic transformation (HT), recanalization status and reperfusion grade at 24 hours, final infarct volume at 7 days and modified Rankin scale (mRS) at 3 months after onset were recorded.

Results

Semi-quantitative and quantitative CTP ASPECTS were highly correlated (p<0.00001). CBF, CBV and MTT ASPECTS were higher in patients with no HT and mRS≤2 and inversely associated with final infarct volume and mRS (p values: from p<0.05 to p<0.00001). CTP ASPECTS mismatch was slightly associated with radiological and clinical outcomes (p values: from p<0.05 to p<0.02) only if evaluated quantitatively. A CBV ASPECTS of 9 was the optimal semi-quantitative value for predicting outcome.

Conclusions

Our findings suggest that visual inspection of CTP ASPECTS recognizes infarct and ischemic absolute values. Semi-quantitative CBV ASPECTS, but not CTP ASPECTS mismatch, represents a strong prognostic indicator, implying that core extent is the main determinant of outcome, irrespective of penumbra size.