Dextromethorphan inhibits collagen and collagen-like cargo secretion to ameliorate lung fibrosis.

Excessive deposition of fibrillar collagen in the interstitial extracellular matrix (ECM) of human lung tissue causes fibrosis, which can ultimately lead to organ failure. Despite our understanding of the molecular mechanisms underlying the disease, no cure for pulmonary fibrosis has yet been found. We screened a drug library and found that dextromethorphan (DXM), a cough expectorant, reduced the amount of excess fibrillar collagen deposited in the ECM in cultured primary human lung fibroblasts, a bleomycin mouse model, and a cultured human precision-cut lung slice model of lung fibrosis.

Palmitoylation of ULK1 by ZDHHC13 plays a crucial role in autophagy.

Autophagy is a highly conserved process from yeast to mammals in which intracellular materials are engulfed by a double-membrane organelle called autophagosome and degrading materials by fusing with the lysosome. The process of autophagy is regulated by sequential recruitment and function of autophagy-related (Atg) proteins. Genetic hierarchical analyses show that the ULK1 complex comprised of ULK1-FIP200-ATG13-ATG101 translocating from the cytosol to autophagosome formation sites as a most upstream ATG factor; this translocation is critical in autophagy initiation.

High-Throughput Microscopy Characterization of Rare Variants.

Familial hypercholesterolemia (FH) is the most common inherited life-threatening disorder of lipid metabolism. Early diagnosis and treatment are the key to reduce the cumulative life-long cardiovascular burden of patients with FH. The high number of variants described as variants of unknown significance is the largest obstacle to achieve a definitive FH diagnosis.

The importance of nuclear RAGE-Mcm2 axis in diabetes or cancer-associated replication stress.

An elevated frequency of DNA replication defects is associated with diabetes and cancer. However, data linking these nuclear perturbations to the onset or progression of organ complications remained unexplored. Here, we report that RAGE (Receptor for Advanced Glycated Endproducts), previously believed to be an extracellular receptor, upon metabolic stress localizes to the damaged forks.

CLEMSite, a software for automated phenotypic screens using light microscopy and FIB-SEM.

In recent years, Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) has emerged as a flexible method that enables semi-automated volume ultrastructural imaging. We present a toolset for adherent cells that enables tracking and finding cells, previously identified in light microscopy (LM), in the FIB-SEM, along with the automatic acquisition of high-resolution volume datasets. We detect the underlying grid pattern in both modalities (LM and EM), to identify common reference points.

Repression of the iron exporter ferroportin may contribute to hepatocyte iron overload in individuals with type 2 diabetes.

Objective: Hyperferremia and hyperferritinemia are observed in patients and disease models of type 2 diabetes mellitus (T2DM). Likewise, patients with genetic iron overload diseases develop diabetes, suggesting a tight link between iron metabolism and diabetes. The liver controls systemic iron homeostasis and is a central organ for T2DM.

Regulation of the COPII secretory machinery via focal adhesions and extracellular matrix signaling.

Proteins that enter the secretory pathway are transported from their place of synthesis in the endoplasmic reticulum to the Golgi complex by COPII-coated carriers. The networks of proteins that regulate these components in response to extracellular cues have remained largely elusive. Using high-throughput microscopy, we comprehensively screened 378 cytoskeleton-associated and related proteins for their functional interaction with the coat protein complex II (COPII) components SEC23A and SEC23B.

Functional profiling of LDLR variants: Important evidence for variant classification: Functional profiling of LDLR variants.

Background: Familial Hypercholesterolemia (FH) is a semidominant disorder of the lipid metabolism associated with premature atherosclerosis and coronary heart disease. So far, about 3,000 unique LDLR variants have been described, most of which lack functional evidence proving their effect on LDLR function, despite the important role that functional studies play in variant classification.

Objective: In this work, we aimed to functionally characterize 13 rare missense variants, identified worldwide and in Portugal, in clinical FH patients.

Systems Approaches to Unravel Molecular Function: High-content siRNA Screen Identifies TMEM16A Traffic Regulators as Potential Drug Targets for Cystic Fibrosis.

An attractive approach to treat people with Cystic Fibrosis (CF), a life-shortening disease caused by mutant CFTR, is to compensate for the absence of this chloride/bicarbonate channel by activating alternative (non-CFTR) chloride channels. One obvious target for such "mutation-agnostic" therapeutic approach is TMEM16A (anoctamin-1/ANO1), a calcium-activated chloride channel (CaCC) which is also expressed in the airways of people with CF, albeit at low levels. To find novel TMEM16A regulators of both traffic and function, with the main goal of identifying candidate CF drug targets, we performed a fluorescence cell-based high-throughput siRNA microscopy screen for TMEM16A trafficking using a double-tagged construct expressed in human airway cells.

A laser-mediated photo-manipulative toolbox for generation and real-time monitoring of DNA lesions.

With the advancement of laser-based microscopy tools, it is now possible to explore mechano-kinetic processes occurring inside the cell. Here, we describe the advanced protocol for studying the DNA repair kinetics in real time using the laser to induce the DNA damage. This protocol can be used for inducing, testing, and studying the repair mechanisms associated with DNA double-strand breaks, interstrand cross-link repair, and single-strand break repair.

Global distribution patterns of marine nitrogen-fixers by imaging and molecular methods.

Nitrogen fixation has a critical role in marine primary production, yet our understanding of marine nitrogen-fixers (diazotrophs) is hindered by limited observations. Here, we report a quantitative image analysis pipeline combined with mapping of molecular markers for mining >2,000,000 images and >1300 metagenomes from surface, deep chlorophyll maximum and mesopelagic seawater samples across 6 size fractions (<0.2-2000 μm).

BigDataProcessor2: a free and open-source Fiji plugin for inspection and processing of TB sized image data.

Summary: Modern bioimaging and related areas such as sensor technology have undergone tremendous development over the last few years. As a result, contemporary imaging techniques, particularly electron microscopy (EM) and light sheet microscopy, can frequently generate datasets attaining sizes of several terabytes (TB). As a consequence, even seemingly simple data operations such as cropping, chromatic- and drift-corrections and even visualisation, poses challenges when applied to thousands of time points or tiles.

An integrated multiomic and quantitative label-free microscopy-based approach to study pro-fibrotic signalling in human precision-cut lung slices.

Fibrosis can affect any organ, resulting in the loss of tissue architecture and function with often life-threatening consequences. Pathologically, fibrosis is characterised by the expansion of connective tissue due to excessive deposition of extracellular matrix (ECM) proteins, including the fibrillar forms of collagen. A significant limitation for discovering cures for fibrosis is the availability of suitable human models and techniques to quantify mature fibrillar collagen deposition as close as possible to human physiological conditions.

Inhibition of calpain 1 restores plasma membrane stability to pharmacologically rescued Phe508del-CFTR variant.

Cystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding CF transmembrane conductance regulator (CFTR), a chloride channel normally expressed at the surface of epithelial cells. The most frequent mutation, resulting in Phe-508 deletion, causes CFTR misfolding and its premature degradation. Low temperature or pharmacological correctors can partly rescue the Phe508del-CFTR processing defect and enhance trafficking of this channel variant to the plasma membrane (PM).

One step ahead: Innovation in core facilities.

Core facilities—originally founded to give scientists access to a specific technology or service—have expanded to become incubators for new technologies and services. While it has many benefits, it also creates unique problems and challenges.[Image: see text]

GM130 and p115 play a key role in the organisation of the early secretory pathway during skeletal muscle differentiation.

Skeletal muscle (SKM) differentiation is a highly regulated process leading to the formation of specialised cells with reorganised compartments and organelles, such as those of the early secretory pathway. During SKM differentiation the Golgi complex (GC) redistributes close to the nuclear envelope and in small distinct peripheral structures distributed throughout the myotube. Concurrently, GC elements closely associate with endoplasmic reticulum-exit sites (ERES).

A novel microscopy-based assay identifies extended synaptotagmin-1 (ESYT1) as a positive regulator of anoctamin 1 traffic.

An attractive possibility to treat Cystic Fibrosis (CF), a severe condition caused by dysfunctional CFTR, an epithelial anion channel, is through the activation of alternative (non-CFTR) anion channels. Anoctamin 1 (ANO1) was demonstrated to be a Ca-activated chloride channel (CaCC) and thus of high potential to replace CFTR. Despite that ANO1 is expressed in human lung CF tissue, it is present at the cell surface at very low levels.

Quantitative 3D-imaging for cell biology and ecology of environmental microbial eukaryotes.

We present a 3D-fluorescence imaging and classification tool for high throughput analysis of microbial eukaryotes in environmental samples. It entails high-content feature extraction that permits accurate automated taxonomic classification and quantitative data about organism ultrastructures and interactions. Using plankton samples from the Oceans expeditions, we validate its applicability to taxonomic profiling and ecosystem analyses, and discuss its potential for future integration of eukaryotic cell biology into evolutionary and ecological studies.

Targeted Ablation Using Laser Nanosurgery.

Laser-mediated dissection methods have been used for many years to micro-irradiate biological samples, but recent technological progress has rendered this technique more precise, powerful, and easy to use. Today pulsed lasers can be operated with diffraction limited, sub-micrometer precision to ablate intracellular structures. Here, we discuss laser nanosurgery setups and the instrumentation in our laboratory.

Profiling DNA damage response following mitotic perturbations.

Genome integrity relies on precise coordination between DNA replication and chromosome segregation. Whereas replication stress attracted much attention, the consequences of mitotic perturbations for genome integrity are less understood. Here, we knockdown 47 validated mitotic regulators to show that a broad spectrum of mitotic errors correlates with increased DNA breakage in daughter cells.

The endosomal transcriptional regulator RNF11 integrates degradation and transport of EGFR.

Stimulation of cells with epidermal growth factor (EGF) induces internalization and partial degradation of the EGF receptor (EGFR) by the endo-lysosomal pathway. For continuous cell functioning, EGFR plasma membrane levels are maintained by transporting newly synthesized EGFRs to the cell surface. The regulation of this process is largely unknown.

Institutional core facilities: prerequisite for breakthroughs in the life sciences: Core facilities play an increasingly important role in biomedical research by providing scientists access to sophisticated technology and expertise.

Core facilities have become an important resource in biomedical research, providing scientists with access to sophisticated instrumentation and expertise. To enable scientists to perform ever more complex and difficult experiments, core facilities not only need to constantly upgrade technology and expertise, but also cooperate and pool their assets. [Image: see text]