11.7T Diffusion Magnetic Resonance Imaging and Tractography to Probe Human Brain Organoid Microstructure.

Background: Human brain organoids are 3-dimensional cellular models that mimic architectural features of a developing brain. Generated from human induced pluripotent stem cells, these organoids offer an unparalleled physiologically relevant in vitro system for disease modeling and drug screening. In the current study, we sought to establish a foundation for a magnetic resonance imaging (MRI)-based, label-free imaging system that offers high-resolution capabilities for deep tissue imaging of whole organoids.

The AKT2/SIRT5/TFEB pathway as a potential therapeutic target in non-neovascular AMD.

Non-neovascular or dry age-related macular degeneration (AMD) is a multi-factorial disease with degeneration of the aging retinal-pigmented epithelium (RPE). Lysosomes play a crucial role in RPE health via phagocytosis and autophagy, which are regulated by transcription factor EB/E3 (TFEB/E3). Here, we find that increased AKT2 inhibits PGC-1α to downregulate SIRT5, which we identify as an AKT2 binding partner.

The AKT2/SIRT5/TFEB pathway as a potential therapeutic target in atrophic AMD.

Age-related macular degeneration (AMD), the leading cause of geriatric blindness, is a multi-factorial disease with retinal-pigmented epithelial (RPE) cell dysfunction as a central pathogenic driver. With RPE degeneration, lysosomal function is a core process that is disrupted. Transcription factors EB/E3 (TFEB/E3) tightly control lysosomal function; their disruption can cause aging disorders, such as AMD.

The Impaired Neurodevelopment of Human Neural Rosettes in HSV-1-Infected Early Brain Organoids.

Intrauterine infections during pregnancy by herpes simplex virus (HSV) can cause significant neurodevelopmental deficits in the unborn/newborn, but clinical studies of pathogenesis are challenging, and while animal models can model some aspects of disease, in vitro studies of human neural cells provide a critical platform for more mechanistic studies. We utilized a reductionist approach to model neurodevelopmental outcomes of HSV-1 infection of neural rosettes, which represent the in vitro equivalent of differentiating neural tubes. Specifically, we employed early-stage brain organoids (ES-organoids) composed of human induced pluripotent stem cells (hiPSCs)-derived neural rosettes to investigate aspects of the potential neuropathological effects induced by the HSV-1 infections on neurodevelopment.

Time-resolved proximity labeling of protein networks associated with ligand-activated EGFR.

Ligand binding to the EGF receptor (EGFR) triggers multiple signal-transduction processes and promotes endocytosis of the receptor. The mechanisms of EGFR endocytosis and its cross-talk with signaling are poorly understood. Here, we combine peroxidase-catalyzed proximity labeling, isobaric peptide tagging, and quantitative mass spectrometry to define the dynamics of the proximity proteome of ligand-activated EGFR.

Increased LCN2 (lipocalin 2) in the RPE decreases autophagy and activates inflammasome-ferroptosis processes in a mouse model of dry AMD.

In dry age-related macular degeneration (AMD), LCN2 (lipocalin 2) is upregulated. Whereas LCN2 has been implicated in AMD pathogenesis, the mechanism remains unknown. Here, we report that in retinal pigmented epithelial (RPE) cells, LCN2 regulates macroautophagy/autophagy, in addition to maintaining iron homeostasis.

Real-time imaging of asthmatic epithelial cells identifies migratory deficiencies under type-2 conditions.

Background: The epithelium is increasingly recognized as a pathologic contributor to asthma and its phenotypes. Although delayed wound closure by asthmatic epithelial cells is consistently observed, underlying mechanisms remain poorly understood, partly due to difficulties in studying dynamic physiologic processes involving polarized multilayered cell systems. Although type-2 immunity has been suggested to play a role, the mechanisms by which repair is diminished are unclear.

Cytoplasmic CPSF6 Regulates HIV-1 Capsid Trafficking and Infection in a Cyclophilin A-Dependent Manner.

Human immunodeficiency virus type 1 (HIV-1) capsid binds host proteins during infection, including cleavage and polyadenylation specificity factor 6 (CPSF6) and cyclophilin A (CypA). We observe that HIV-1 infection induces higher-order CPSF6 formation, and capsid-CPSF6 complexes cotraffic on microtubules. CPSF6-capsid complex trafficking is impacted by capsid alterations that reduce CPSF6 binding or by excess cytoplasmic CPSF6 expression, both of which are associated with decreased HIV-1 infection.

Direct coupling of oligomerization and oligomerization-driven endocytosis of the dopamine transporter to its conformational mechanics and activity.

Dopamine transporter (DAT) mediates the reuptake of synaptically released dopamine, and thus controls the duration and intensity of dopamine neurotransmission. Mammalian DAT has been observed to form oligomers, although the mechanisms of oligomerization and its role in DAT activity and trafficking remain largely unknown. We discovered a series of small molecule compounds that stabilize trimers and induce high-order oligomers of DAT and concomitantly promote its clathrin-independent endocytosis.

PLA2G6 guards placental trophoblasts against ferroptotic injury.

The recently identified ferroptotic cell death is characterized by excessive accumulation of hydroperoxy-arachidonoyl (C20:4)- or adrenoyl (C22:4)- phosphatidylethanolamine (Hp-PE). The selenium-dependent glutathione peroxidase 4 (GPX4) inhibits ferroptosis, converting unstable ferroptotic lipid hydroperoxides to nontoxic lipid alcohols in a tissue-specific manner. While placental oxidative stress and lipotoxicity are hallmarks of placental dysfunction, the possible role of ferroptosis in placental dysfunction is largely unknown.

Regulation of ALT-associated homology-directed repair by polyADP-ribosylation.

The synthesis of poly(ADP-ribose) (PAR) reconfigures the local chromatin environment and recruits DNA-repair complexes to damaged chromatin. PAR degradation by poly(ADP-ribose) glycohydrolase (PARG) is essential for progression and completion of DNA repair. Here, we show that inhibition of PARG disrupts homology-directed repair (HDR) mechanisms that underpin alternative lengthening of telomeres (ALT).

Live-cell imaging and analysis reveal cell phenotypic transition dynamics inherently missing in snapshot data.

Recent advances in single-cell techniques catalyze an emerging field of studying how cells convert from one phenotype to another, in a step-by-step process. Two grand technical challenges, however, impede further development of the field. Fixed cell-based approaches can provide snapshots of high-dimensional expression profiles but have fundamental limits on revealing temporal information, and fluorescence-based live-cell imaging approaches provide temporal information but are technically challenging for multiplex long-term imaging.

Autocrine C-peptide protects INS1 β cells against palmitic acid-induced oxidative stress in peroxisomes by inducing catalase.

Aims: C-peptide, produced by pancreatic β cells and co-secreted in the bloodstream with insulin, has antioxidant properties in glucose- and hydrogen peroxide (HO)-exposed INS1 β cells. Palmitic acid, the most physiologically abundant long-chain free fatty acid in humans, is metabolized in peroxisomes of β cells accumulating HO that can lead to oxidative stress. Here, we tested the hypothesis that C-peptide protects β cells from palmitic acid-induced stress by lowering peroxisomal HO.

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.

Neurokinin-1 Receptor Signaling Is Required for Efficient Ca Flux in T-Cell-Receptor-Activated T Cells.

Efficient Ca flux induced during cognate T cell activation requires signaling the T cell receptor (TCR) and unidentified G-protein-coupled receptors (GPCRs). T cells express the neurokinin-1 receptor (NK1R), a GPCR that mediates Ca flux in excitable and non-excitable cells. However, the role of the NK1R in TCR signaling remains unknown.

RAD51AP1 Is an Essential Mediator of Alternative Lengthening of Telomeres.

Alternative lengthening of telomeres (ALT) is a homology-directed repair (HDR) mechanism of telomere elongation that controls proliferation in aggressive cancers. We show that the disruption of RAD51-associated protein 1 (RAD51AP1) in ALT+ cancer cells leads to generational telomere shortening. This is due to RAD51AP1's involvement in RAD51-dependent homologous recombination (HR) and RAD52-POLD3-dependent break induced DNA synthesis.

The Lysophosphatidylcholine Transporter MFSD2A Is Essential for CD8 Memory T Cell Maintenance and Secondary Response to Infection.

Access to nutrients is critical for an effective T cell immune response to infection. Although transporters for sugars and amino acids have previously been described in the context of the CD8 T cell immune response, the active transport of exogenous fatty acids has remained enigmatic. In this study, we discovered that the sodium-dependent lysophosphatidylcholine (LPC) transporter major facilitator superfamily domain containing 2A (MFSD2A) is upregulated on activated CD8 T cells and is required for memory T cell maintenance.

Learn to segment single cells with deep distance estimator and deep cell detector.

Single cell segmentation is a critical and challenging step in cell imaging analysis. Traditional processing methods require time and labor to manually fine-tune parameters and lack parameter transferability between different situations. Recently, deep convolutional neural networks (CNN) treat segmentation as a pixel-wise classification problem and have become a general and efficient method for image segmentation.

Mitochondria are a substrate of cellular memory.

Cellular memory underlies cellular identity, and thus constitutes a unifying mechanism of genetic disposition, environmental influences, and cellular adaptation. Here, we demonstrate that enduring physicochemical changes of mitochondrial networks invoked by transient stress, a phenomenon we term 'mitoengrams', underlie the transgenerational persistence of epigenetically scripted cellular behavior. Using C2C12 myogenic stem-like cells, we show that stress memory elicited by transient, low-level arsenite exposure is stored within a self-renewing subpopulation of progeny cells in a mitochondrial-dependent fashion.

Compensatory increases of select proteostasis networks after Hsp70 inhibition in cancer cells.

Cancer cells thrive when challenged with proteotoxic stress by inducing components of the protein folding, proteasome, autophagy and unfolded protein response (UPR) pathways. Consequently, specific molecular chaperones have been validated as targets for anti-cancer therapies. For example, inhibition of Hsp70 family proteins (hereafter Hsp70) in rhabdomyosarcoma triggers UPR induction and apoptosis.

Chloroquine reduces hypercoagulability in pancreatic cancer through inhibition of neutrophil extracellular traps.

Background: The hypercoagulable state associated with pancreatic adenocarcinoma (PDA) results in increased risk of venous thromboembolism, leading to substantial morbidity and mortality. Recently, neutrophil extracellular traps (NETs), whereby activated neutrophils release their intracellular contents containing DNA, histones, tissue factor, high mobility group box 1 (HMGB1) and other components have been implicated in PDA and in cancer-associated thrombosis.

Methods: Utilizing an orthotopic murine PDA model in C57/Bl6 mice and patient correlative samples, we studied the role of NETs in PDA hypercoagulability and targeted this pathway through treatment with the NET inhibitor chloroquine.