An aldehyde-crosslinking mitochondrial probe for STED imaging in fixed cells.

Fluorescence labeling of chemically fixed specimens, especially immunolabeling, plays a vital role in super-resolution imaging as it offers a convenient way to visualize cellular structures like mitochondria or the distribution of biomolecules with high detail. Despite the development of various distinct probes that enable super-resolved stimulated emission depletion (STED) imaging of mitochondria in live cells, most of these membrane-potential-dependent fluorophores cannot be retained well in mitochondria after chemical fixation. This lack of suitable mitochondrial probes has limited STED imaging of mitochondria to live cell samples. In this study, we introduce a mitochondria-specific probe, PK Mito Orange FX (PKMO FX), which features a fixation-driven cross-linking motif and accumulates in the mitochondrial inner membrane. It exhibits high fluorescence retention after chemical fixation and efficient depletion at 775 nm, enabling nanoscopic imaging both before and after aldehyde fixation. We demonstrate the compatibility of this probe with conventional immunolabeling and other strategies commonly used for fluorescence labeling of fixed samples. Moreover, we show that PKMO FX facilitates correlative super-resolution light and electron microscopy, enabling the correlation of multicolor fluorescence images and transmission EM images via the characteristic mitochondrial pattern. Our probe further expands the mitochondrial toolkit for multimodal microscopy at nanometer resolutions.

Enhanced mitochondrial fusion during a critical period of synaptic plasticity in adult-born neurons.

Integration of new neurons into adult hippocampal circuits is a process coordinated by local and long-range synaptic inputs. To achieve stable integration and uniquely contribute to hippocampal function, immature neurons are endowed with a critical period of heightened synaptic plasticity, yet it remains unclear which mechanisms sustain this form of plasticity during neuronal maturation. We found that as new neurons enter their critical period, a transient surge in fusion dynamics stabilizes elongated mitochondrial morphologies in dendrites to fuel synaptic plasticity. Conditional ablation of fusion dynamics to prevent mitochondrial elongation selectively impaired spine plasticity and synaptic potentiation, disrupting neuronal competition for stable circuit integration, ultimately leading to decreased survival. Despite profuse mitochondrial fragmentation, manipulation of competition dynamics was sufficient to restore neuronal survival but left neurons poorly responsive to experience at the circuit level. Thus, by enabling synaptic plasticity during the critical period, mitochondrial fusion facilitates circuit remodeling by adult-born neurons.

Correction: Winter et al. Z-Disk-Associated Plectin (Isoform 1d): Spatial Arrangement, Interaction Partners, and Role in Filamin C Homeostasis. Cells 2023, 12, 1259.

The authors wish to make the following changes to their paper [...].

Docking and stability defects in mitofusin highlight the proteasome as a potential therapeutic target.

Defects in mitochondrial fusion are at the base of many diseases. Mitofusins power membrane-remodeling events via self-interaction and GTP hydrolysis. However, how exactly mitofusins mediate fusion of the outer membrane is still unclear. Structural studies enable tailored design of mitofusin variants, providing valuable tools to dissect this stepwise process. Here, we found that the two cysteines conserved between yeast and mammals are required for mitochondrial fusion, revealing two novel steps of the fusion cycle. C381 is dominantly required for the formation of the trans-tethering complex, before GTP hydrolysis. C805 allows stabilizing the Fzo1 protein and the trans-tethering complex, just prior to membrane fusion. Moreover, proteasomal inhibition rescued Fzo1 C805S levels and membrane fusion, suggesting a possible application for clinically approved drugs. Together, our study provides insights into how assembly or stability defects in mitofusins might cause mitofusin-associated diseases and uncovers potential therapeutic intervention by proteasomal inhibition.

Z-Disk-Associated Plectin (Isoform 1d): Spatial Arrangement, Interaction Partners, and Role in Filamin C Homeostasis.

Plectin, a highly versatile cytolinker protein, is crucial for myofiber integrity and function. Accordingly, mutations in the human gene (PLEC) cause several rare diseases, denoted as plectinopathies, with most of them associated with progressive muscle weakness. Of several plectin isoforms expressed in skeletal muscle and the heart, P1d is the only isoform expressed exclusively in these tissues. Using high-resolution stimulated emission depletion (STED) microscopy, here we show that plectin is located within the gaps between individual α-actinin-positive Z-disks, recruiting and bridging them to desmin intermediate filaments (Ifs). Loss of plectin in myofibril bundles led to a complete loss of desmin Ifs. Loss of Z-disk-associated plectin isoform P1d led to disorganization of muscle fibers and slower relaxation of myofibrils upon mechanical strain, in line with an observed inhomogeneity of muscle ultrastructure. In addition to binding to α-actinin and thereby providing structural support, P1d forms a scaffolding platform for the chaperone-assisted selective autophagy machinery (CASA) by directly interacting with HSC70 and synpo2. In isoform-specific knockout (P1d-KO) mouse muscle and mechanically stretched plectin-deficient myoblasts, we found high levels of undigested filamin C, a bona fide substrate of CASA. Similarly, subjecting P1d-KO mice to forced swim tests led to accumulation of filamin C aggregates in myofibers, highlighting a specific role of P1d in tension-induced proteolysis activated upon high loads of physical exercise and muscle contraction.

Localization of Mitochondrial Nucleoids by Transmission Electron Microscopy Using the Transgenic Expression of the Mitochondrial Helicase Twinkle and APEX2.

Reminiscent of their evolutionary origin, mitochondria contain their own genome (mtDNA) compacted into the mitochondrial chromosome or nucleoid (mt-nucleoid). Many mitochondrial disorders are characterized by disruption of mt-nucleoids, either by direct mutation of genes involved in mtDNA organization or by interfering with other vital proteins for mitochondrial function. Thus, changes in mt-nucleoid morphology, distribution, and structure are a common feature in many human diseases and can be exploited as an indicator of cellular fitness. Electron microscopy provides the highest possible resolution that can be achieved, delivering spatial and structural information about all cellular structures. Recently, the ascorbate peroxidase APEX2 has been used to increase transmission electron microscopy (TEM) contrast by inducing diaminobenzidine (DAB) precipitation. DAB has the ability to accumulate osmium during classical EM sample preparation and, due to its high electron density, provides strong contrast for TEM. Among the nucleoid proteins, the mitochondrial helicase Twinkle fused with APEX2 has been successfully used to target mt-nucleoids, providing a tool to visualize these subcellular structures with high contrast and with the resolution of an electron microscope. In the presence of H2O2, APEX2 catalyzes the polymerization of DAB, generating a brown precipitate that can be visualized in specific regions of the mitochondrial matrix. Here, we provide a detailed protocol to generate murine cell lines expressing a transgenic variant of Twinkle, suitable to target and visualize mt-nucleoids. We also describe all the necessary steps to validate the cell lines prior to electron microscopy imaging and offer examples of anticipated results.

Capsazepine (CPZ) Inhibits TRPC6 Conductance and Is Protective in Adriamycin-Induced Nephropathy and Diabetic Glomerulopathy.

Reactive oxygen species (ROS), which excessively arise in diabetes and systemic inflammatory diseases, modify cellular lipids and cellular lipid composition leading to altered biophysical properties of cellular membranes. The impact of lipid peroxidation on transmembrane signaling routes is not yet well studied. The canonical transient receptor potential channel 6 (TRPC6) is implicated in the pathogenesis of several forms of glomerular diseases. TRPC6 is sensitive to membrane stretch and relies on a distinct lipid environment. This study investigates the effect of oxidative alterations to plasma membrane lipids on TRPC6 activity and the function of the glomerular filter. Knockout of the anti-oxidative, lipid modifying enzyme paraoxonase 2 (PON2) leads to altered biophysical properties of glomerular epithelial cells, which are called podocytes. Cortical stiffness, quantified by atomic force microscopy, was largely increased in PON2-deficient cultured podocytes. PON2 deficiency markedly enhanced TRPC6 channel currents and channel recovery. Treatment with the amphiphilic substance capsazepine in micromolar doses reduced cortical stiffness and abrogated TRPC6 conductance. In in vivo studies, capsazepine reduced the glomerular phenotype in the model of adriamycin-induced nephropathy in PON2 knockout mice and wildtype littermates. In diabetic AKITA mice, the progression of albuminuria and diabetic kidney disease was delayed. In summary, we provide evidence that the modification of membrane characteristics affects TRPC6 signaling. These results could spur future research to investigate modification of the direct lipid environment of TRPC6 as a future therapeutic strategy in glomerular disease.

Application of the adverse outcome pathway concept for investigating developmental neurotoxicity potential of Chinese herbal medicines by using human neural progenitor cells in vitro.

Adverse outcome pathways (AOPs) are organized sequences of key events (KEs) that are triggered by a xenobiotic-induced molecular initiating event (MIE) and summit in an adverse outcome (AO) relevant to human or ecological health. The AOP framework causally connects toxicological mechanistic information with apical endpoints for application in regulatory sciences. AOPs are very useful to link endophenotypic, cellular endpoints in vitro to adverse health effects in vivo. In the field of in vitro developmental neurotoxicity (DNT), such cellular endpoints can be assessed using the human "Neurosphere Assay," which depicts different endophenotypes for a broad variety of neurodevelopmental KEs. Combining this model with large-scale transcriptomics, we evaluated DNT hazards of two selected Chinese herbal medicines (CHMs) Lei Gong Teng (LGT) and Tian Ma (TM), and provided further insight into their modes-of-action (MoA). LGT disrupted hNPC migration eliciting an exceptional migration endophenotype. Time-lapse microscopy and intervention studies indicated that LGT disturbs laminin-dependent cell adhesion. TM impaired oligodendrocyte differentiation in human but not rat NPCs and activated a gene expression network related to oxidative stress. The LGT results supported a previously published AOP on radial glia cell adhesion due to interference with integrin-laminin binding, while the results of TM exposure were incorporated into a novel putative, stressor-based AOP. This study demonstrates that the combination of phenotypic and transcriptomic analyses is a powerful tool to elucidate compounds' MoA and incorporate the results into novel or existing AOPs for a better perception of the DNT hazard in a regulatory context.

Mitochondrial membrane proteins and VPS35 orchestrate selective removal of mtDNA.

Understanding the mechanisms governing selective turnover of mutation-bearing mtDNA is fundamental to design therapeutic strategies against mtDNA diseases. Here, we show that specific mtDNA damage leads to an exacerbated mtDNA turnover, independent of canonical macroautophagy, but relying on lysosomal function and ATG5. Using proximity labeling and Twinkle as a nucleoid marker, we demonstrate that mtDNA damage induces membrane remodeling and endosomal recruitment in close proximity to mitochondrial nucleoid sub-compartments. Targeting of mitochondrial nucleoids is controlled by the ATAD3-SAMM50 axis, which is disrupted upon mtDNA damage. SAMM50 acts as a gatekeeper, influencing BAK clustering, controlling nucleoid release and facilitating transfer to endosomes. Here, VPS35 mediates maturation of early endosomes to late autophagy vesicles where degradation occurs. In addition, using a mouse model where mtDNA alterations cause impairment of muscle regeneration, we show that stimulation of lysosomal activity by rapamycin, selectively removes mtDNA deletions without affecting mtDNA copy number, ameliorating mitochondrial dysfunction. Taken together, our data demonstrates that upon mtDNA damage, mitochondrial nucleoids are eliminated outside the mitochondrial network through an endosomal-mitophagy pathway. With these results, we unveil the molecular players of a complex mechanism with multiple potential benefits to understand mtDNA related diseases, inherited, acquired or due to normal ageing.

CD30-Positive Extracellular Vesicles Enable the Targeting of CD30-Negative DLBCL Cells by the CD30 Antibody-Drug Conjugate Brentuximab Vedotin.

CD30, a member of the TNF receptor superfamily, is selectively expressed on a subset of activated lymphocytes and on malignant cells of certain lymphomas, such as classical Hodgkin Lymphoma (cHL), where it activates critical bystander cells in the tumor microenvironment. Therefore, it is not surprising that the CD30 antibody-drug conjugate Brentuximab Vedotin (BV) represents a powerful, FDA-approved treatment option for CD30+ hematological malignancies. However, BV also exerts a strong anti-cancer efficacy in many cases of diffuse large B cell lymphoma (DLBCL) with poor CD30 expression, even when lacking detectable CD30+ tumor cells. The mechanism remains enigmatic. Because CD30 is released on extracellular vesicles (EVs) from both, malignant and activated lymphocytes, we studied whether EV-associated CD30 might end up in CD30- tumor cells to provide binding sites for BV. Notably, CD30+ EVs bind to various DLBCL cell lines as well as to the FITC-labeled variant of the antibody-drug conjugate BV, thus potentially conferring the BV binding also to CD30- cells. Confocal microscopy and imaging cytometry studies revealed that BV binding and uptake depend on CD30+ EVs. Since BV is only toxic toward CD30- DLBCL cells when CD30+ EVs support its uptake, we conclude that EVs not only communicate within the tumor microenvironment but also influence cancer treatment. Ultimately, the CD30-based BV not only targets CD30+ tumor cell but also CD30- DLBCL cells in the presence of CD30+ EVs. Our study thus provides a feasible explanation for the clinical impact of BV in CD30- DLBCL and warrants confirming studies in animal models.

Extraocular Muscle Reveals Selective Vulnerability of Type IIB Fibers to Respiratory Chain Defects Induced by Mitochondrial DNA Alterations.

Purpose: The purpose of this study was to gain insights on the pathogenesis of chronic progressive external ophthalmoplegia, thus we investigated the vulnerability of five extra ocular muscles (EOMs) fiber types to pathogenic mitochondrial DNA deletions in a mouse model expressing a mutated mitochondrial helicase TWINKLE. Methods: Consecutive pairs of EOM sections were analyzed by cytochrome C oxidase (COX)/succinate dehydrogenase (SDH) assay and fiber type specific immunohistochemistry (type I, IIA, IIB, embryonic, and EOM-specific staining). Results: The mean average of COX deficient fibers (COX-) in the recti muscles of mutant mice was 1.04 ± 0.52% at 12 months and increased with age (7.01 ± 1.53% at 24 months). A significant proportion of these COX- fibers were of the fast-twitch, glycolytic type IIB (> 50% and > 35% total COX- fibers at 12 and 24 months, respectively), whereas embryonic myosin heavy chain-expressing fibers were almost completely spared. Furthermore, the proportion of COX- fibers in the type IIB-rich retractor bulbi muscle was > 2-fold higher compared to the M. recti at both 12 (2.6 ± 0.78%) and 24 months (20.85 ± 2.69%). Collectively, these results demonstrate a selective vulnerability of type IIB fibers to mitochondrial DNA (mtDNA) deletions in EOMs and retractor bulbi muscle. We also show that EOMs of mutant mice display histopathological abnormalities, including altered fiber type composition, increased fibrosis, ragged red fibers, and infiltration of mononucleated nonmuscle cells. Conclusions: Our results point to the existence of fiber type IIB-intrinsic factors and/or molecular mechanisms that predispose them to increased generation, clonal expansion, and detrimental effects of mtDNA deletions.

Microtubule-dependent and independent roles of spastin in lipid droplet dispersion and biogenesis.

Lipid droplets (LDs) are metabolic organelles that store neutral lipids and dynamically respond to changes in energy availability by accumulating or mobilizing triacylglycerols (TAGs). How the plastic behavior of LDs is regulated is poorly understood. Hereditary spastic paraplegia is a central motor axonopathy predominantly caused by mutations in SPAST, encoding the microtubule-severing protein spastin. The spastin-M1 isoform localizes to nascent LDs in mammalian cells; however, the mechanistic significance of this targeting is not fully explained. Here, we show that tightly controlled levels of spastin-M1 are required to inhibit LD biogenesis and TAG accumulation. Spastin-M1 maintains the morphogenesis of the ER when TAG synthesis is prevented, independent from microtubule binding. Moreover, spastin plays a microtubule-dependent role in mediating the dispersion of LDs from the ER upon glucose starvation. Our results reveal a dual role of spastin to shape ER tubules and to regulate LD movement along microtubules, opening new perspectives for the pathogenesis of hereditary spastic paraplegia.

Mitochondria-Endoplasmic Reticulum Contacts in Reactive Astrocytes Promote Vascular Remodeling.

Astrocytes have emerged for playing important roles in brain tissue repair; however, the underlying mechanisms remain poorly understood. We show that acute injury and blood-brain barrier disruption trigger the formation of a prominent mitochondrial-enriched compartment in astrocytic endfeet, which enables vascular remodeling. Integrated imaging approaches revealed that this mitochondrial clustering is part of an adaptive response regulated by fusion dynamics. Astrocyte-specific conditional deletion of Mitofusin 2 (Mfn2) suppressed perivascular mitochondrial clustering and disrupted mitochondria-endoplasmic reticulum (ER) contact sites. Functionally, two-photon imaging experiments showed that these structural changes were mirrored by impaired mitochondrial Ca2+ uptake leading to abnormal cytosolic transients within endfeet in vivo. At the tissue level, a compromised vascular complexity in the lesioned area was restored by boosting mitochondrial-ER perivascular tethering in MFN2-deficient astrocytes. These data unmask a crucial role for mitochondrial dynamics in coordinating astrocytic local domains and have important implications for repairing the injured brain.

Mitochondrial Dysfunction Combined with High Calcium Load Leads to Impaired Antioxidant Defense Underlying the Selective Loss of Nigral Dopaminergic Neurons.

Mitochondrial dysfunction is critically involved in Parkinson's disease, characterized by loss of dopaminergic neurons (DaNs) in the substantia nigra (SNc), whereas DaNs in the neighboring ventral tegmental area (VTA) are much less affected. In contrast to VTA, SNc DaNs engage calcium channels to generate action potentials, which lead to oxidant stress by yet unknown pathways. To determine the molecular mechanisms linking calcium load with selective cell death in the presence of mitochondrial deficiency, we analyzed the mitochondrial redox state and the mitochondrial membrane potential in mice of both sexes with genetically induced, severe mitochondrial dysfunction in DaNs (MitoPark mice), at the same time expressing a redox-sensitive GFP targeted to the mitochondrial matrix. Despite mitochondrial insufficiency in all DaNs, exclusively SNc neurons showed an oxidized redox-system, i.e., a low reduced/oxidized glutathione (GSH-GSSG) ratio. This was mimicked by cyanide, but not by rotenone or antimycin A, making the involvement of reactive oxygen species rather unlikely. Surprisingly, a high mitochondrial inner membrane potential was maintained in MitoPark SNc DaNs. Antagonizing calcium influx into the cell and into mitochondria, respectively, rescued the disturbed redox ratio and induced further hyperpolarization of the inner mitochondrial membrane. Our data therefore show that the constant calcium load in SNc DaNs is counterbalanced by a high mitochondrial inner membrane potential, even under conditions of severe mitochondrial dysfunction, but triggers a detrimental imbalance in the mitochondrial redox system, which will lead to neuron death. Our findings thus reveal a new mechanism, redox imbalance, which underlies the differential vulnerability of DaNs to mitochondrial defects.SIGNIFICANCE STATEMENT Parkinson's disease is characterized by the preferential degeneration of dopaminergic neurons (DaNs) of the substantia nigra pars compacta (SNc), resulting in the characteristic hypokinesia in patients. Ubiquitous pathological triggers cannot be responsible for the selective neuron loss. Here we show that mitochondrial impairment together with elevated calcium burden destabilize the mitochondrial antioxidant defense only in SNc DaNs, and thus promote the increased vulnerability of this neuron population.

Injured Podocytes Are Sensitized to Angiotensin II-Induced Calcium Signaling.

BACKGROUND: Inhibition of angiotensin II (AngII) signaling, a therapeutic mainstay of glomerular kidney diseases, is thought to act primarily through regulating glomerular blood flow and reducing filtration pressure. Although extravascular actions of AngII have been suggested, a direct effect of AngII on podocytes has not been demonstrated in vivo. METHODS: To study the effects of AngII on podocyte calcium levels in vivo, we used intravital microscopy of the kidney in mice expressing the calcium indicator protein GCaMP3. RESULTS: In healthy animals, podocytes displayed limited responsiveness to AngII stimulation. In contrast, in animals subjected to either adriamycin-induced acute chemical injury or genetic deletion of the podocin-encoding gene Nphs2, the consequent podocyte damage and proteinuria rendered the cells responsive to AngII and resulted in AngII-induced calcium transients in significantly more podocytes. The angiotensin type 1 receptor blocker losartan could fully inhibit this response. Also, responsiveness to AngII was at least partly mediated through the transient receptor potential channel 6, which has been implicated in podocyte calcium handling. Interestingly, loss of a single Nphs2 allele also increased podocytes' responsiveness to AngII signaling. This direct effect of AngII on injured podocytes results in increased calcium transients, which can further aggravate the underlying kidney disease. CONCLUSIONS: Our discovery that podocytes become sensitized to AngII-induced calcium signaling upon injury might explain results from large, randomized, controlled trials in which improved renal outcomes occur only in the subgroup of patients with proteinuria, indicating podocyte damage. Our findings also emphasize the need to treat every patient with a glomerular disease with either an angiotensin-converting enzyme inhibitor or an angiotensin type 1 receptor blocker.

Effect of Maternal Obesity in Mice on IL-6 Levels and Placental Endothelial Cell Homeostasis.

Obesity during pregnancy is a known health risk for mother and child. Since obesity is associated with increased inflammatory markers, our objectives were to determine interleukin-6 (IL-6) levels in obese mice and to examine the effect of IL-6 on placental endothelial cells. Placentas, blood, and adipose tissue of C57BL/6N mice, kept on high fat diet before and during pregnancy, were harvested at E15.5. Serum IL-6 levels were determined and endothelial cell markers and IL-6 expression were measured by qRT-PCR and western blot. Immunostaining was used to determine surface and length densities of fetal capillary profiles and placental endothelial cell homeostasis. Human placental vein endothelial cells were cultured and subjected to proliferation, apoptosis, senescence, and tube formation assays after stimulation with hyperIL-6. Placental endothelial cell markers were downregulated and the percentage of senescent endothelial cells was higher in the placental exchange zone of obese dams and placental vascularization was strongly reduced. Additionally, maternal IL-6 serum levels and IL-6 protein levels in adipose tissue were increased. Stimulation with hyperIL-6 provoked a dose dependent increase of senescence in cultured endothelial cells without any effects on proliferation or apoptosis. Diet-induced maternal obesity led to an IUGR phenotype accompanied by increased maternal IL-6 serum levels. In the placenta of obese dams, this may result in a disturbed endothelial cell homeostasis and impaired fetal vasculature. Cell culture experiments confirmed that IL-6 is capable of inducing endothelial cell senescence.

Plasticity in salt bridge allows fusion-competent ubiquitylation of mitofusins and Cdc48 recognition.

Mitofusins are dynamin-related GTPases that drive mitochondrial fusion by sequential events of oligomerization and GTP hydrolysis, followed by their ubiquitylation. Here, we show that fusion requires a trilateral salt bridge at a hinge point of the yeast mitofusin Fzo1, alternatingly forming before and after GTP hydrolysis. Mutations causative of Charcot-Marie-Tooth disease massively map to this hinge point site, underlining the disease relevance of the trilateral salt bridge. A triple charge swap rescues the activity of Fzo1, emphasizing the close coordination of the hinge residues with GTP hydrolysis. Subsequently, ubiquitylation of Fzo1 allows the AAA-ATPase ubiquitin-chaperone Cdc48 to resolve Fzo1 clusters, releasing the dynamin for the next fusion round. Furthermore, cross-complementation within the oligomer unexpectedly revealed ubiquitylated but fusion-incompetent Fzo1 intermediates. However, Cdc48 did not affect the ubiquitylated but fusion-incompetent variants, indicating that Fzo1 ubiquitylation is only controlled after membrane merging. Together, we present an integrated model on how mitochondrial outer membranes fuse, a critical process for their respiratory function but also putatively relevant for therapeutic interventions.

Mould-reactive T cells for the diagnosis of invasive mould infection-A prospective study.

Invasive mould infections (IMI) in immunocompromised patients are difficult to diagnose. Early and targeted treatment is paramount, but minimally invasive tests reliably identifying pathogens are lacking. We previously showed that monitoring pathogen-specific CD4+T cells in peripheral blood using upregulation of induced CD154 positive lymphocytes can be used to diagnose acute IMI. Here, we validate our findings in an independent patient cohort. We stimulated peripheral blood cells from at-risk patients with Aspergillus spp. and Mucorales lysates and quantitated mould-reactive CD4/CD69/CD154 positive lymphocytes via flow cytometry. Mould-reactive lymphocytes were quantitated in 115 at-risk patients. In 38 (33%) patients, the test was not evaluable, mainly due to low T cell counts or non-reactive positive control. Test results were evaluable in 77 (67%) patients. Of these, four patients (5%) had proven IMI and elevated mould-reactive T cell signals. Of 73 (95%) patients without proven IMI, 59 (81%) had mould-reactive T cell signals within normal range. Fourteen (19%) patients without confirmed IMI showed elevated T cell signals and 11 of those received antifungal treatment. The mould-reactive lymphocyte assay identified presence of IMI with a sensitivity of 100% and specificity of 81%. The mould-reactive lymphocyte assay correctly identified all patients with proven IMI. Assay applicability is limited by low T cell counts during bone marrow suppression. The assay has the potential to support diagnosis of invasive mould infection to facilitate tailored treatment even when biopsies are contraindicated or cultures remain negative.

Extracellular vesicle measurements with nanoparticle tracking analysis - An accuracy and repeatability comparison between NanoSight NS300 and ZetaView.

The expanding field of extracellular vesicle (EV) research needs reproducible and accurate methods to characterize single EVs. Nanoparticle Tracking Analysis (NTA) is commonly used to determine EV concentration and diameter. As the EV field is lacking methods to easily confirm and validate NTA data, questioning the reliability of measurements remains highly important. In this regard, a comparison addressing measurement quality between different NTA devices such as Malvern's NanoSight NS300 or Particle Metrix' ZetaView has not yet been conducted. To evaluate the accuracy and repeatability of size and concentration determinations of both devices, we employed comparative methods including transmission electron microscopy (TEM) and single particle interferometric reflectance imaging sensing (SP-IRIS) by ExoView. Multiple test measurements with nanospheres, liposomes and ultracentrifuged EVs from human serum and cell culture supernatant were performed. Additionally, serial dilutions and freeze-thaw cycle-dependent EV decrease were measured to determine the robustness of each system. Strikingly, NanoSight NS300 exhibited a 2.0-2.1-fold overestimation of polystyrene and silica nanosphere concentration. By measuring serial dilutions of EV samples, we demonstrated higher accuracy in concentration determination by ZetaView (% BIAS range: 2.7-8.5) in comparison with NanoSight NS300 (% BIAS range: 32.9-36.8). The concentration measurements by ZetaView were also more precise (% CV range: 0.0-4.7) than measurements by NanoSight NS300 (% CV range: 5.4-10.7). On the contrary, quantitative TEM imaging indicated more accurate EV sizing by NanoSight NS300 (% DTEM range: 79.5-134.3) compared to ZetaView (% DTEM range: 111.8-205.7), while being equally repeatable (NanoSight NS300% CV range: 0.8-6.7; ZetaView: 1.4-7.8). However, both devices failed to report a peak EV diameter below 60 nm compared to TEM and SP-IRIS. Taken together, NTA devices differ strongly in their hardware and software affecting measuring results. ZetaView provided a more accurate and repeatable depiction of EV concentration, whereas NanoSight NS300 supplied size measurements of higher resolution.

Selective Capture and Purification of MicroRNAs and Intracellular Proteins through Antisense-vectorized Magnetic Nanobeads.

MicroRNAs (miRNAs) are small non-coding nucleotides playing a crucial role in posttranscriptional expression and regulation of target genes in nearly all kinds of cells. In this study, we demonstrate a reliable and efficient capture and purification of miRNAs and intracellular proteins using magnetic nanoparticles functionalized with antisense oligonucleotides. For this purpose, a tumor suppressor miRNA (miR-198), deregulated in several human cancer types, was chosen as the model oligonucleotide. Magnetite nanoparticles carrying the complementary sequence of miR-198 (miR-198 antisense) on their surface were delivered into cells and subsequently used for the extracellular transport of miRNA and proteins. The successful capture of miR-198 was demonstrated by isolating RNA from magnetic nanoparticles followed by real-time PCR quantification. Our experimental data showed that antisense-coated particles captured 5-fold higher amounts of miR-198 when compared to the control nanoparticles. Moreover, several proteins that could play a significant role in miR-198 biogenesis were found attached to miR-198 conjugated nanoparticles and analyzed by mass spectrometry. Our findings demonstrate that a purpose-driven vectorization of magnetic nanobeads with target-specific recognition ligands is highly efficient in selectively transporting miRNA and disease-relevant proteins out of cells and could become a reliable and useful tool for future diagnostic, therapeutic and analytical applications.