Metarhizium anisopliae, a fungal pathogen of terrestrial arthropods, kills the aquatic larvae of Aedes aegypti, the vector of dengue and yellow fever. The fungus kills without adhering to the host cuticle. Ingested conidia also fail to germinate and are expelled in fecal pellets. This study investigates the mechanism by which this fungus adapted to terrestrial hosts kills aquatic mosquito larvae. Genes associated with the M. anisopliae early pathogenic response (proteinases Pr1 and Pr2, and adhesins, Mad1 and Mad2) are upregulated in the presence of larvae, but the established infection process observed in terrestrial hosts does not progress and insecticidal destruxins were not detected. Protease inhibitors reduce larval mortality indicating the importance of proteases in the host interaction. The Ae. aegypti immune response to M. anisopliae appears limited, whilst the oxidative stress response gene encoding for thiol peroxidase is upregulated. Cecropin and Hsp70 genes are downregulated as larval death occurs, and insect mortality appears to be linked to autolysis through caspase activity regulated by Hsp70 and inhibited, in infected larvae, by protease inhibitors. Evidence is presented that a traditional host-pathogen response does not occur as the species have not evolved to interact. M. anisopliae retains pre-formed pathogenic determinants which mediate host mortality, but unlike true aquatic fungal pathogens, does not recognise and colonise the larval host.

White blood cell (WBC) differential counting is an established clinical routine to assess patient immune system status. Fluorescent markers and a flow cytometer are required for the current state‐of‐the‐art method for determining WBC differential counts. However, this process requires several sample preparation steps and may adversely disturb the cells. We present a novel label‐free approach using an imaging flow cytometer and machine learning algorithms, where live, unstained WBCs were classified. It achieved an average F1‐score of 97% and two subtypes of WBCs, B and T lymphocytes, were distinguished from each other with an average F1‐score of 78%, a task previously considered impossible for unlabeled samples. We provide an open‐source workflow to carry out the procedure. We validated the WBC analysis with unstained samples from 85 donors. The presented method enables robust and highly accurate identification of WBCs, minimizing the disturbance to the cells and leaving marker channels free to answer other biological questions. It also opens the door to employing machine learning for liquid biopsy, here, using the rich information in cell morphology for a wide range of diagnostics of primary blood. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Hard corona (HC) protein, i.e., the environmental proteins of the biological medium that are bound to a nanosurface, is known to affect the biological fate of a nanomedicine. Due to the size, curvature, and specific surface area (SSA) 3-factor interactions inherited in the traditional 3D nanoparticle, HC-dependent bio-nano interactions are often poorly probed and interpreted. Here, the first HC-by-design case study in 2D is demonstrated that sequentially and linearly changes the HC quantity using functionalized graphene oxide (GO) nanosheets. The HC quantity and HC quality are analyzed using NanoDrop and label-free liquid chromatography-mass spectrometry (LC-MS) followed by principal component analysis (PCA). Cellular responses (uptake and cytotoxicity in J774 cell model) are compared using imaging cytometry and the modified lactate dehydrogenase assays, respectively. Cellular uptake linearly and solely correlates with HC quantity (R = 0.99634). The nanotoxicity, analyzed by retrospective design of experiment (DoE), is found to be dependent on the nanomaterial uptake (primary), HC composition (secondary), and nanomaterial exposure dose (tertiary). This unique 2D design eliminates the size-curvature-SSA multifactor interactions and can serve as a reliable screening platform to uncover HC-dependent bio-nano interactions to enable the next-generation quality-by-design (QbD) nanomedicines for better clinical translation.

SummaryLeucocytes respond rapidly to pathogenic and other insults, with responses ranging from cytokine production to migration and phagocytosis. These are bioenergetically expensive, and increased glycolytic flux provides adenosine triphosphate (ATP) rapidly to support these essential functions. However, much of this work is from animal studies. To understand more clearly the relative role of glycolysis and oxidative phosphorylation in human leucocytes, especially their utility in a translational research setting, we undertook a study of human peripheral blood mononuclear cells (MNCs) bioenergetics. Glycolysis was essential during lipopolysaccharide (LPS)-mediated interleukin (IL)21b, IL-6 and tumour necrosis factor (TNF)-a production, as 2-deoxy-D-glucose decreased significantly the output of all three cytokines. After optimizing cell numbers and the concentrations of all activators and inhibitors, oxidative phosphorylation and glycolysis profiles of fresh and cryopreserved/resuscitated MNCs were determined to explore the utility of MNCs for determining the bioenergetics health profile in multiple clinical settings. While the LPS-induced cytokine response did not differ significantly between fresh and resuscitated cells from the same donors, cryopreservation/resuscitation significantly affected mainly some measures of oxidative phosphorylation, but also glycolysis. Bioenergetics analysis of human MNCs provides a quick, effective means to measure the bioenergetics health index of many individuals, but cryopreserved cells are not suitable for such an analysis. The translational utility of this approach was tested by comparing MNCs of pregnant and non-pregnant women to reveal increased bioenergetics health index with pregnancy but significantly reduced basal glycolysis and glycolytic capacity. More detailed analysis of discrete leucocyte populations would be required to understand the relative roles of glycolysis and oxidative phosphorylation during inflammation and other immune responses.

We report on a pedigree with a pair of brothers each with minor anomalies, developmental delay and autistic-symptoms who share an unbalanced translocation (not detectable by karyotype). The unbalanced translocation involves a 7.1 Mb loss of the terminal portion of 10q, and a 4.2Mb gain of 11q. One of the brothers also developed a cerebellar juvenile pilocytic astrocytoma. The father was found to be a balanced carrier and the couple had a previous miscarriage. We demonstrate that the breakpoint for the triplicated region from chromosome 11 is adjacent to two IgLON genes, namely Neurotrimin (NTM) and Opioid Binding/Cell Adhesion Molecule-like (OPCML). These genes are highly similar neural cell adhesion molecules that have been implicated in synaptogenesis and oncogenesis respectively. The children also have a 10q deletion and are compared to other children with the 10q deletion syndrome which generally does not involve autism spectrum disorders or cancer. Together these data support a role for NTM and OPCML in developmental delay and potentially in cancer susceptibility.