Neuropsychiatric Aspects of Infectious Diseases: An Update

Among the critically ill, infectious diseases can play a significant role in the etiology of neuropsychiatric disturbances. All critical care physicians are familiar with delirium as a secondary complication of systemic infection. This article focuses on key infectious diseases that commonly and directly produce neuropsychiatric symptoms, including direct infection of the central nervous system, human immunodeficiency virus infection, and AIDS

Development of a Rapid Insulin Assay by Homogenous Time-Resolved Fluorescence

Direct measurement of insulin is critical for basic and clinical studies of insulin secretion. However, current methods are expensive and time-consuming. We developed an insulin assay based on homogenous time-resolved fluorescence that is significantly more rapid and cost-effective than current commonly used approaches. This assay was applied effectively to an insulin secreting cell line, INS-1E cells, as well as pancreatic islets, allowing us to validate the assay by elucidating mechanisms by which dopamine regulates insulin release. We found that dopamine functioned as a significant negative modulator of glucose-stimulated insulin secretion. Further, we showed that bromocriptine, a known dopamine D2/D3 receptor agonist and newly approved drug used for treatment of type II diabetes mellitus, also decreased glucose-stimulated insulin secretion in islets to levels comparable to those caused by dopamine treatment

Distinguishing Signal From Autofluorescence In Cryogenic Correlated Light And Electron Microscopy Of Mammalian Cells

In cryogenic correlated light and electron microscopy (cryo-CLEM), frozen targets of interest are identified and located on EM grids by fluorescence microscopy and then imaged at higher resolution by cryo-EM. Whilst working with these methods, we discovered that a variety of mammalian cells exhibit strong punctate autofluorescence when imaged under cryogenic conditions (80K). Autofluorescence originated from multilamellar bodies (MLBs) and secretory granules. Here we describe a method to distinguish fluorescent protein tags from these autofluorescent sources based on the narrower emission spectrum of the former. The method is first tested on mitochondria and then applied to examine the ultrastructural variability of secretory granules within insulin-secreting pancreatic beta-cell-derived INS-1E cells

Mechanisms of amphetamine action illuminated through optical monitoring of dopamine synaptic vesicles in Drosophila brain

Amphetamines elevate extracellular dopamine, but the underlying mechanisms remain uncertain. Here we show in rodents that acute pharmacological inhibition of the vesicular monoamine transporter (VMAT) blocks amphetamine-induced locomotion and self-administration without impacting cocaine-induced behaviours. To study VMAT’s role in mediating amphetamine action in dopamine neurons, we have used novel genetic, pharmacological and optical approaches in Drosophila melanogaster. In an ex vivo whole-brain preparation, fluorescent reporters of vesicular cargo and of vesicular pH reveal that amphetamine redistributes vesicle contents and diminishes the vesicle pH-gradient responsible for dopamine uptake and retention. This amphetamine-induced deacidification requires VMAT function and results from net H+ antiport by VMAT out of the vesicle lumen coupled to inward amphetamine transport. Amphetamine-induced vesicle deacidification also requires functional dopamine transporter (DAT) at the plasma membrane. Thus, we find that at pharmacologically relevant concentrations, amphetamines must be actively transported by DAT and VMAT in tandem to produce psychostimulant effects

Detecting anomalies from liquid transfer videos in automated laboratory setting

In this work, we address the problem of detecting anomalies in a certain laboratory automation setting. At first, we collect video images of liquid transfer in automated laboratory experiments. We mimic the real-world challenges of developing an anomaly detection model by considering two points. First, the size of the collected dataset is set to be relatively small compared to large-scale video datasets. Second, the dataset has a class imbalance problem where the majority of the collected videos are from abnormal events. Consequently, the existing learning-based video anomaly detection methods do not perform well. To this end, we develop a practical human-engineered feature extraction method to detect anomalies from the liquid transfer video images. Our simple yet effective method outperforms state-of-the-art anomaly detection methods with a notable margin. In particular, the proposed method provides 19% and 76% average improvement in AUC and Equal Error Rate, respectively. Our method also quantifies the anomalies and provides significant benefits for deployment in the real-world experimental setting

Three-dimensional analysis of mitochondrial crista ultrastructure in a Leigh Syndrome patient by in situ cryo-electron tomography

Mitochondrial diseases produce profound neurological dysfunction via mutations affecting mitochondrial energy production, including the relatively common Leigh Syndrome (LS). We recently described an LS case caused by a pathogenic mutation in USMG5, encoding a small supernumerary subunit of mitochondrial ATP synthase. This protein is integral for ATP synthase dimerization, and patient fibroblasts revealed an almost total loss of ATP synthase dimers. Here, we utilize in situ cryo-electron tomography (cryo-ET) in a clinical case-control study of mitochondrial disease to directly study mitochondria within cultured fibroblasts from an LS patient and a healthy human control subject. Through tomographic analysis of patient and control mitochondria, we find that loss of ATP synthase dimerization caused by the pathogenic mutation causes profound disturbances of mitochondrial crista ultrastructure. Overall, this work supports the crucial role of ATP synthase in regulating crista architecture in the context of human disease

Ribosome-associated vesicles: A dynamic subcompartment of the endoplasmic reticulum in secretory cells

The endoplasmic reticulum (ER) is a highly dynamic network of membranes. Here, we combine live-cell microscopy with in situ cryo–electron tomography to directly visualize ER dynamics in several secretory cell types including pancreatic β-cells and neurons under near-native conditions. Using these imaging approaches, we identify a novel, mobile form of ER, ribosome-associated vesicles (RAVs), found primarily in the cell periphery, which is conserved across different cell types and species. We show that RAVs exist as distinct, highly dynamic structures separate from the intact ER reticular architecture that interact with mitochondria via direct intermembrane contacts. These findings describe a new ER subcompartment within cells