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Over 80% of the patients with pancreatic ductal adenocarcinoma (PDAC) have cachexia/wasting syndrome. Cachexia is associated with reduced survival, decreased quality of life, and higher metastasis rates. Here, we demonstrate that fat loss is the earliest feature of PDAC-exosome-induced cachexia. MicroRNA sequencing of exosomal components from normal and cancer-derived exosomes revealed enrichment of miR-16-5p, miR-21-5p, miR-29a-3p, and miR-125b-5p in serum exosomes of mice harboring PDAC and patients with PDAC. Further, miR-16-5p and miR-29a-3p inhibited adipogenesis through decreasing Erlin2 and Cmpk1 expression which downregulates C/EBPβ and PPARγ. Synergistically, miR-29a-3p promotes lipolysis through increasing ATGL expression by suppressing MCT1 expression. Furthermore, PDAC-exosomes deprived of miR-16-5p and miR-29a-3p fail to induce fat loss. Hence, miR-16-5p and miR-29a-3p exosomal miRs are essential for PDAC-induced fat loss. Thus, we unravel that PDAC induces adipose atrophy via exosomal miRs. This knowledge may provide new diagnostic and therapeutic strategies for PDAC-induced cachexia.

Fertilization occurs before the completion of oocyte meiosis in the majority of animal species and sperm contents move long distances within the zygotes of mouse and C. elegans. If incorporated into the meiotic spindle, paternal chromosomes could be expelled into a polar body resulting in lethal monosomy. Through live imaging of fertilization in C. elegans, we found that the microtubule disassembling enzymes, katanin and kinesin-13 limit long-range movement of sperm contents and that maternal ataxin-2 maintains paternal DNA and paternal mitochondria as a cohesive unit that moves together. Depletion of katanin or double depletion of kinesin-13 and ataxin-2 resulted in the capture of the sperm contents by the meiotic spindle. Thus limiting movement of sperm contents and maintaining cohesion of sperm contents within the zygote both contribute to preventing premature interaction between maternal and paternal genomes.

Healthy adipose tissue is essential for normal physiology. There are 2 broad types of adipose tissue depots: brown adipose tissue (BAT), which contains adipocytes poised to burn energy through thermogenesis, and white adipose tissue (WAT), which contains adipocytes that store lipids. However, within those types of adipose, adipocytes possess depot and cell-specific properties that have important implications. For example, the subcutaneous and visceral WAT confers divergent risk for metabolic disease. Further, within a depot, different adipocytes can have distinct properties; subcutaneous WAT can contain adipocytes with either white or brown-like (beige) adipocyte properties. However, the pathways that regulate and maintain this cell and depot-specificity are incompletely understood. Here, we found that the transcription factor KLF15 is required for maintaining white adipocyte properties selectively within the subcutaneous WAT. We revealed that deletion of Klf15 is sufficient to induce beige adipocyte properties and that KLF15s direct regulation of Adrb1 is a critical molecular mechanism for this process. We uncovered that this activity is cell autonomous but has systemic implications in mouse models and is conserved in primary human adipose cells. Our results elucidate a pathway for depot-specific maintenance of white adipocyte properties that could enable the development of therapies for obesity and associated diseases.

Recent studies demonstrate that liver secretory proteins, also known as hepatokines, regulate normal development, obesity, and simple steatosis to non-alcoholic steatohepatitis (NASH) progression. Using a panel of ∼100 diverse inbred strains of mice and a cohort of bariatric surgery patients, we found that one such hepatokine, inter-trypsin inhibitor heavy chain 3 (ITIH3), was progressively lower in severe non-alcoholic fatty liver disease (NAFLD) disease states highlighting an inverse relationship between Itih3/ITIH3 expression and NAFLD severity. Follow-up animal and cell culture models demonstrated that hepatic ITIH3 overexpression lowered liver triglyceride and lipid droplet accumulation, respectively. Conversely, ITIH3 knockdown in mice increased the liver triglyceride in two independent NAFLD models. Mechanistically, ITIH3 reduced mitochondrial respiration and this, in turn, reduced liver triglycerides, via downregulated de novo lipogenesis. This was accompanied by increased STAT1 signaling and Stat3 expression, both of which are known to protect against NAFLD/NASH. Our findings indicate hepatokine ITIH3 as a potential biomarker and/or treatment for NAFLD.

Deep learning is transforming bioimage analysis, but its application in single-cell segmentation is limited by the lack of large, diverse annotated datasets. We addressed this by introducing a CycleGAN-based architecture, cGAN-Seg, that enhances the training of cell segmentation models with limited annotated datasets. During training, cGAN-Seg generates annotated synthetic phase-contrast or fluorescent images with morphological details and nuances closely mimicking real images. This increases the variability seen by the segmentation model, enhancing the authenticity of synthetic samples and thereby improving predictive accuracy and generalization. Experimental results show that cGAN-Seg significantly improves the performance of widely used segmentation models over conventional training techniques. Our approach has the potential to accelerate the development of foundation models for microscopy image analysis, indicating its significance in advancing bioimage analysis with efficient training methodologies.

Nuclear factor kappa B (NF-κB) is a key regulator in immune signaling and is known to exhibit a digital activation pattern. Yet the molecular basis underlying the heterogeneity in NF-κB activation at single-cell level is not entirely understood. Here, we show that NF-κB activation in single cells is largely regulated by intrinsic differences at the receptor level. Using the genome editing and time-lapse imaging, we directly characterize endogenous TNFR1 dynamics and NF-κB activation from the same single cells. Total internal reflection fluorescence (TIRF) microscopy shows that endogenous TNFR1 forms pre-ligand clusters in the resting cells. Upon tumor necrosis factor (TNF) stimulation, the diffusion coefficient of membrane TNFR1 was significantly decreased and a substantial level of TNFR1 undergoes oligomerization to form trimers and hexamers. Moreover, multi-color cell imaging reveals that both digital and graded information processing regulate NF-κB activation across different TNFR1 expression levels. Our results indicate that single-cell NF-κB activation potential strongly correlates with its TNFR1 characteristics.

Fragile X syndrome (FXS) is caused by the loss of fragile X messenger ribonucleoprotein (FMRP), a translational regulator that binds the transcripts of proteins involved in synaptic function and plasticity. Dysregulated protein synthesis is a central effect of FMRP loss, however, direct translational modulation has not been leveraged in the treatment of FXS. Thus, we examined the effect of the translational modulator integrated stress response inhibitor (ISRIB) in treating synaptic and behavioral symptoms of FXS. We show that FMRP loss dysregulates synaptic protein abundance, stabilizing dendritic spines through increased PSD-95 levels while preventing spine maturation through reduced glutamate receptor accumulation, thus leading to the formation of dense, immature dendritic spines, characteristic of FXS patients and Fmr1 knockout (KO) mice. ISRIB rescues these deficits and improves social recognition in Fmr1 KO mice. These findings highlight the therapeutic potential of targeting core translational mechanisms in FXS and neurodevelopmental disorders more broadly.

Facioscapulohumeral dystrophy (FSHD) is linked to contraction of D4Z4 repeats on chromosome 4q with SMCHD1 mutations acting as a disease modifier. D4Z4 heterochromatin disruption and abnormal upregulation of the transcription factor DUX4, encoded in the D4Z4 repeat, are the hallmarks of FSHD. However, defining the precise effect of D4Z4 contraction has been difficult because D4Z4 repeats are primate-specific and DUX4 expression is very rare in highly heterogeneous patient myocytes. We generated isogenic mutant cell lines harboring D4Z4 and/or SMCHD1 mutations in a healthy human skeletal myoblast line. We found that the mutations affect D4Z4 heterochromatin differently, and that SMCHD1 mutation or disruption of DNA methylation stabilizes otherwise variegated DUX4 target activation in D4Z4 contraction mutant cells, demonstrating the critical role of modifiers. Our study revealed amplification of the DUX4 signal through downstream targets, H3.X/Y and LEUTX. Our results provide important insights into how rare DUX4 expression leads to FSHD pathogenesis.

The RNA-binding protein PARP13 is a primary factor in the innate antiviral response, which suppresses translation and drives decay of bound viral and host RNA. PARP13 interacts with many proteins encoded by interferon-stimulated genes (ISG) to activate antiviral pathways including co-translational addition of ISG15, or ISGylation. We performed enhanced crosslinking immunoprecipitation (eCLIP) and RNA-seq in human cells to investigate PARP13's role in transcriptome regulation for both basal and antiviral states. We find that the antiviral response shifts PARP13 target localization, but not its binding preferences, and that PARP13 supports the expression of ISGylation-related genes, including PARP13's cofactor, TRIM25. PARP13 associates with TRIM25 via RNA-protein interactions, and we elucidate a transcriptome-wide periodicity of PARP13 binding around TRIM25. Taken together, our study implicates PARP13 in creating and maintaining a cellular environment poised for an antiviral response through limiting PARP13 translation, regulating access to distinct mRNA pools, and elevating ISGylation machinery expression.

In Arabidopsis thaliana, ARGONAUTE1 (AGO1) plays a central role in microRNA (miRNA) and small interfering RNA (siRNA)-mediated silencing. AGO1 associates to the rough endoplasmic reticulum to conduct miRNA-mediated translational repression, mRNA cleavage, and biogenesis of phased siRNAs. Here, we show that a 37°C heat stress (HS) promotes AGO1 protein accumulation in cytosolic condensates where it colocalizes with components of siRNA bodies and of stress granules. AGO1 contains a prion-like domain in its poorly characterized N-terminal Poly-Q domain, which is sufficient to undergo phase separation independently of the presence of SGS3. HS only moderately affects the small RNA repertoire, the loading of AGO1 by miRNAs, and the signatures of target cleavage, suggesting that its localization in condensates protects AGO1 rather than promoting or impairing its activity in reprogramming gene expression during stress. Collectively, our work sheds new light on the impact of high temperature on a main effector of RNA silencing in plants.

High-grade serous carcinoma (HGSC) is the most lethal gynecological malignancy in the United States. Late diagnosis and the emergence of chemoresistance have prompted studies into how the tumor microenvironment, and more recently tumor innervation, may be leveraged for HGSC prevention and interception. In addition to stess-induced sources, concentrations of the sympathetic neurotransmitter norepinephrine (NE) in the ovary increase during ovulation and after menopause. Importantly, NE exacerbates advanced HGSC progression. However, little is known about the role of NE in early disease pathogenesis. Here, we investigated the role of NE in instigating anchorage independence and micrometastasis of preneoplastic lesions from the fallopian tube epithelium (FTE) to the ovary, an essential step in HGSC onset. We found that in the presence of NE, FTE cell lines were able to survive in ultra-low-attachment (ULA) culture in a β-adrenergic receptor-dependent (β-AR-dependent) manner. Importantly, spheroid formation and cell viability conferred by treatment with physiological sources of NE were abrogated using the β-AR blocker propranolol. We have also identified that NE-mediated anoikis resistance may be attributable to downregulation of colony-stimulating factor 2. These findings provide mechanistic insight and identify targets that may be regulated by ovary-derived NE in early HGSC.

Vascular calcification is a severe complication of cardiovascular diseases. Previous studies demonstrated that endothelial lineage cells transitioned into osteoblast-like cells and contributed to vascular calcification. Here, we found that inhibition of cyclin-dependent kinase (CDK) prevented endothelial lineage cells from transitioning to osteoblast-like cells and reduced vascular calcification. We identified a robust induction of CDK1 in endothelial cells (ECs) in calcified arteries and showed that EC-specific gene deletion of CDK1 decreased the calcification. We found that limiting CDK1 induced E-twenty-six specific sequence variant 2 (ETV2), which was responsible for blocking endothelial lineage cells from undergoing osteoblast differentiation. We also found that inhibition of CDK1 reduced vascular calcification in a diabetic mouse model. Together, the results highlight the importance of CDK1 suppression and suggest CDK1 inhibition as a potential option for treating vascular calcification.

Interferon (IFN) activity exhibits a gender bias in human skin, skewed toward females. We show that HERC6, an IFN-induced E3 ubiquitin ligase, is induced in human keratinocytes through the epidermal type I IFN; IFN-κ. HERC6 knockdown in human keratinocytes results in enhanced induction of interferon-stimulated genes (ISGs) upon treatment with a double-stranded (ds) DNA STING activator cGAMP but not in response to the RNA-sensing TLR3 agonist. Keratinocytes lacking HERC6 exhibit sustained STING-TBK1 signaling following cGAMP stimulation through modulation of LATS2 and TBK1 activity, unmasking more robust ISG responses in female keratinocytes. This enhanced female-biased immune response with loss of HERC6 depends on VGLL3, a regulator of type I IFN signature. These data identify HERC6 as a previously unrecognized negative regulator of ISG expression specific to dsDNA sensing and establish it as a regulator of female-biased immune responses through modulation of STING signaling.

Olaparib is a pioneering PARP inhibitor (PARPi) approved for treating castration-resistant prostate cancer (CRPC) tumors harboring DNA repair defects, but clinical resistance has been documented. To study acquired resistance, we developed Olaparib-resistant (OlapR) cell lines through chronic Olaparib treatment of LNCaP and C4-2B cell lines. Here, we found that IGFBP3 is highly expressed in acquired (OlapR) and intrinsic (Rv1) models of Olaparib resistance. We show that IGFBP3 expression promotes Olaparib resistance by enhancing DNA repair capacity through activation of EGFR and DNA-PKcs. IGFBP3 depletion enhances efficacy of Olaparib by promoting DNA damage accumulation and subsequently, cell death in resistant models. Mechanistically, we show that silencing IGFBP3 or EGFR expression reduces cell viability and resensitizes OlapR cells to Olaparib treatment. Inhibition of EGFR by Gefitinib suppressed growth of OlapR cells and improved Olaparib sensitivity, thereby phenocopying IGFBP3 inhibition. Collectively, our results highlight IGFBP3 and EGFR as critical mediators of Olaparib resistance.

Mitochondrial membrane potential directly powers many critical functions of mitochondria, including ATP production, mitochondrial protein import, and metabolite transport. Its loss is a cardinal feature of aging and mitochondrial diseases, and cells closely monitor membrane potential as an indicator of mitochondrial health. Given its central importance, it is logical that cells would modulate mitochondrial membrane potential in response to demand and environmental cues, but there has been little exploration of this question. We report that loss of the Sit4 protein phosphatase in yeast increases mitochondrial membrane potential, both by inducing the electron transport chain and the phosphate starvation response. Indeed, a similarly elevated mitochondrial membrane potential is also elicited simply by phosphate starvation or by abrogation of the Pho85-dependent phosphate sensing pathway. This enhanced membrane potential is primarily driven by an unexpected activity of the ADP/ATP carrier. We also demonstrate that this connection between phosphate limitation and enhancement of mitochondrial membrane potential is observed in primary and immortalized mammalian cells as well as in Drosophila. These data suggest that mitochondrial membrane potential is subject to environmental stimuli and intracellular signaling regulation and raise the possibility for therapeutic enhancement of mitochondrial function even in defective mitochondria.

Successful genome editing in primary human islets could reveal features of the genetic regulatory landscape underlying β cell function and diabetes risk. Here, we describe a CRISPR-based strategy to interrogate functions of predicted regulatory DNA elements using electroporation of a complex of Cas9 ribonucleoprotein (Cas9 RNP) and guide RNAs into primary human islet cells. We successfully targeted coding regions including the PDX1 exon 1, and non-coding DNA linked to diabetes susceptibility. CRISPR-Cas9 RNP approaches revealed genetic targets of regulation by DNA elements containing candidate diabetes risk SNPs, including an in vivo enhancer of the MPHOSPH9 gene. CRISPR-Cas9 RNP multiplexed targeting of two cis-regulatory elements linked to diabetes risk in PCSK1, which encodes an endoprotease crucial for Insulin processing, also demonstrated efficient simultaneous editing of PCSK1 regulatory elements, resulting in impaired β cell PCSK1 regulation and Insulin secretion. Multiplex CRISPR-Cas9 RNP provides powerful approaches to investigate and elucidate human islet cell gene regulation in health and diabetes.

Cholera is a global health problem with no targeted therapies. The Ca2+-sensing receptor (CaSR) is a regulator of intestinal ion transport and a therapeutic target for diarrhea, and Ca2+ is considered its main agonist. We found that increasing extracellular Ca2+ had a minimal effect on forskolin-induced Cl- secretion in human intestinal epithelial T84 cells. However, extracellular Mg2+, an often-neglected CaSR agonist, suppressed forskolin-induced Cl- secretion in T84 cells by 65% at physiological levels seen in stool (10 mM). The effect of Mg2+ occurred via the CaSR/Gq signaling that led to cAMP hydrolysis. Mg2+ (10 mM) also suppressed Cl- secretion induced by cholera toxin, heat-stable E. coli enterotoxin, and vasoactive intestinal peptide by 50%. In mouse intestinal closed loops, luminal Mg2+ treatment (20 mM) inhibited cholera toxin-induced fluid accumulation by 40%. In a mouse intestinal perfusion model of cholera, addition of 10 mM Mg2+ to the perfusate reversed net fluid transport from secretion to absorption. These results suggest that Mg2+ is the key CaSR activator in mouse and human intestinal epithelia at physiological levels in stool. Since stool Mg2+ concentrations in patients with cholera are essentially zero, oral Mg2+ supplementation, alone or in an oral rehydration solution, could be a potential therapy for cholera and other cyclic nucleotide-mediated secretory diarrheas.

The use of patient-derived organoids (PDOs) to characterize therapeutic sensitivity and resistance is a promising precision medicine approach, and its potential to inform clinical decisions is now being tested in several large multiinstitutional clinical trials. PDOs are cultivated in the extracellular matrix from basement membrane extracts (BMEs) that are most commonly acquired commercially. Each clinical site utilizes distinct BME lots and may be restricted due to the availability of commercial BME sources. However, the effect of different sources of BMEs on organoid drug response is unknown. Here, we tested the effect of BME source on proliferation, drug response, and gene expression in mouse and human pancreatic ductal adenocarcinoma (PDA) organoids. Both human and mouse organoids displayed increased proliferation in Matrigel compared with Cultrex and UltiMatrix. However, we observed no substantial effect on drug response when organoids were cultured in Matrigel, Cultrex, or UltiMatrix. We also did not observe major shifts in gene expression across the different BME sources, and PDOs maintained their classical or basal-like designation. Overall, we found that the BME source (Matrigel, Cultrex, UltiMatrix) does not shift PDO dose-response curves or drug testing results, indicating that PDO pharmacotyping is a robust approach for precision medicine.

While the involvement of actin polymerization in cell migration is well-established, much less is known about the role of transmembrane water flow in cell motility. Here, we investigate the role of water influx in a prototypical migrating cell, the neutrophil, which undergoes rapid, directed movement to sites of injury, and infection. Chemoattractant exposure both increases cell volume and potentiates migration, but the causal link between these processes are not known. We combine single-cell volume measurements and a genome-wide CRISPR screen to identify the regulators of chemoattractant-induced neutrophil swelling, including NHE1, AE2, PI3K-gamma, and CA2. Through NHE1 inhibition in primary human neutrophils, we show that cell swelling is both necessary and sufficient for the potentiation of migration following chemoattractant stimulation. Our data demonstrate that chemoattractant-driven cell swelling complements cytoskeletal rearrangements to enhance migration speed.

Pro-preadipocytes are adipocyte progenitor cells within subcutaneous adipose tissue that are conserved in human adipose tissue with distinct cellular energetics. Here, we detail a protocol to quantify cellular oxygen consumption rates of primary human cells harvested from adipose tissue. We describe steps for primary cell expansion, cell seeding, transfection, differentiation, and respirometry followed by Agilent Seahorse Analytics. The measurement of bioenergetic profiles and resulting data further expand our knowledge of the functional properties of primary cells isolated from adipose tissue. For complete details on the use and execution of this protocol, please refer to Chen et al. (2023).1.

Recent advances in computing power triggered the use of artificial intelligence in image analysis in life sciences. To train these algorithms, a large enough set of certified labeled data is required. The trained neural network is then capable of producing accurate instance segmentation results that will then need to be re‐assembled into the original dataset: the entire process requires substantial expertise and time to achieve quantifiable results. To speed‐up the process, from cell organelle detection to quantification across electron microscopy modalities, we propose a deep‐learning based approach for fast automatic outline segmentation (FAMOUS), that involves organelle detection combined with image morphology, and 3D meshing to automatically segment, visualize and quantify cell organelles within volume electron microscopy datasets. From start to finish, FAMOUS provides full segmentation results within a week on previously unseen datasets. FAMOUS was showcased on a HeLa cell dataset acquired using a focused ion beam scanning electron microscope, and on yeast cells acquired by transmission electron tomography. Research Highlights: Introducing a rapid, multimodal machine‐learning workflow for the automatic segmentation of 3D cell organelles.Successfully applied to a variety of volume electron microscopy datasets and cell lines.Outperforming manual segmentation methods in time and accuracy.Enabling high‐throughput quantitative cell biology. [ABSTRACT FROM AUTHOR]

My path to becoming a scientist has taken many twists and turns. This is perhaps not unusual to hear. Indeed, in discussions with my colleagues it seems that for many of us the path was never a straight one. Certainly, for me there have been moments when my whole world was encompassed by science and at other times, I have felt strongly that my time in science was up. I like to think that as scientists we ask a lot of questions and, for many of us, those questions extend to our very purpose as a scientist. My intention with this article is not to document my career path in detail or to provide very specific advice. Rather, I hope to describe how questions have defined my journey and to inspire others to occasionally pause and ask themselves what a career in science means to them. Today, I am an Assistant Professor at a major Canadian university, and here are the questions I asked along the way. [ABSTRACT FROM AUTHOR]

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Hertwig's rule states that cells divide along their longest axis, usually driven by forces acting on the mitotic spindle. Here, we show that in contrast to this rule, microtubule-based pulling forces in early Caenorhabditis elegans embryos align the spindle with the short axis of the cell. We combine theory with experiments to reveal that in order to correct this misalignment, inward forces generated by the constricting cytokinetic ring rotate the entire cell until the spindle is aligned with the cell's long axis. Experiments with slightly compressed mouse zygotes indicate that this cytokinetic ring-driven mechanism of ensuring Hertwig's rule is general for cells capable of rotating inside a confining shell, a scenario that applies to early cell divisions of many systems. [ABSTRACT FROM AUTHOR]

Collective cell migration, where cells move as a cohesive unit, is a vital process underlying morphogenesis and cancer metastasis. Thanks to recent advances in imaging and modelling, we are beginning to understand the intricate relationship between a cell and its microenvironment and how this shapes cell polarity, metabolism and modes of migration. The use of biophysical and mathematical models offers a fresh perspective on how cells migrate collectively, either flowing in a fluid-like state or transitioning to more static states. Continuing to unite researchers in biology, physics and mathematics will enable us to decode more complex biological behaviours that underly collective cell migration; only then can we understand how this coordinated movement of cells influences the formation and organisation of tissues and directs the spread of metastatic cancer. In this Perspective, we highlight exciting discoveries, emerging themes and common challenges that have arisen in recent years, and possibleways forward to bridge the gaps in our current understanding of collective cell migration. [ABSTRACT FROM AUTHOR]

The primary cilium, an antenna-like sensory organelle that protrudes from the surface of most eukaryotic cell types, has become a signaling hub of growing interest given that defects in its structure and/or function are associated with human diseases and syndromes, known as ciliopathies. With the continuously expanding role of primary cilia in health and diseases, identifying new players in ciliogenesis will lead to a better understanding of the function of this organelle. It has been shown that the primary cilium shares similarities with the immune synapse, a highly organized structure at the interface between an antigen-presenting or target cell and a lymphocyte. Studies have demonstrated a role for known cilia regulators in immune synapse formation. However, whether immune synapse regulators modulate ciliogenesis remains elusive. Here, we find that programmed death ligand 1 (PD-L1), an immune checkpoint protein and regulator of immune synapse formation, plays a role in the regulation of ciliogenesis. We found that PD-L1 is enriched at the centrosome/basal body and Golgi apparatus of ciliated cells and depleting PD-L1 enhanced ciliogenesis and increased the accumulation of ciliary membrane trafficking proteins Rab8a, BBS5, and sensory receptor protein PC-2. Moreover, PD-L1 formed a complex with BBS5 and PC-2. In addition, we found that depletion of PD-L1 resulted in the ciliary accumulation of Gli3 and the downregulation of Gli1. Our results suggest that PD-L1 is a new player in ciliogenesis, contributing to PC-2-mediated sensory signaling and the Hh signaling cascade. [ABSTRACT FROM AUTHOR]

Background: Petasites japonicus (PJ), also known as Butterbur, has a rich history as a traditional healing remedy across numerous countries. Objectives: This study was designed to evaluate the potential anti-cancer properties of the methanol extract derived from PJ (PJE). Materials and Methods: Cell viability was measured with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. Cell cycle analysis, caspase activity assays, western blotting, and reactive oxygen species (ROS) assays were also used to investigate the anticancer effects of PJE on cancer cells. Results: It was shown that PJE inhibited the cell viability of the colon carcinoma cell line Caco-2 (half-maximal inhibitory concentration [IC50]: 268.4 µg/mL), of the hepatocellular carcinoma cell line Hep3B (IC50: 420.2 µg/mL), and of the bladder carcinoma cell line 5637 (IC50: 99.43 µg/mL). Analysis of DNA content indicated an increase in the sub-G1 population of 5637 cells as a result of PJE treatment. Furthermore, PJE caused a reduction in mitochondrial membrane potential and the ratio of Bcl-2 to Bax. Moreover, PJE enhanced the levels of various components involved in the proapoptotic cascade, such as caspase-3, caspase-9, and poly-adenosine diphosphate-ribose polymerase. Moreover, it was observed that PJE modulated mitogen-activated protein kinases (MAPKs) activation and induced an elevation in intracellular production of ROS. Conclusion: These combined results strongly suggest that PJE possesses significant proapoptotic properties as an herbal medicine, acting through ROS-dependent MAPK signaling pathways in bladder cancer cells. [ABSTRACT FROM AUTHOR]

Antibodies are critical reagents to detect and characterize proteins. It is commonly understood that many commercial antibodies do not recognize their intended targets, but information on the scope of the problem remains largely anecdotal, and as such, feasibility of the goal of at least one potent and specific antibody targeting each protein in a proteome cannot be assessed. Focusing on antibodies for human proteins, we have scaled a standardized characterization approach using parental and knockout cell lines (Laflamme et al., 2019) to assess the performance of 614 commercial antibodies for 65 neuroscience-related proteins. Side-by-side comparisons of all antibodies against each target, obtained from multiple commercial partners, have demonstrated that: (i) more than 50% of all antibodies failed in one or more applications, (ii) yet, ~50-75% of the protein set was covered by at least one high-performing antibody, depending on application, suggesting that coverage of human proteins by commercial antibodies is significant; and (iii) recombinant antibodies performed better than monoclonal or polyclonal antibodies. The hundreds of underperforming antibodies identified in this study were found to have been used in a large number of published articles, which should raise alarm. Encouragingly, more than half of the underperforming commercial antibodies were reassessed by the manufacturers, and many had alterations to their recommended usage or were removed from the market. This first study helps demonstrate the scale of the antibody specificity problem but also suggests an efficient strategy toward achieving coverage of the human proteome; mine the existing commercial antibody repertoire, and use the data to focus new renewable antibody generation efforts.

Small open reading frame (smORF)-encoded microproteins, proteins containing less than 100-150 amino acids, are an emerging class of functional biomolecules. Here, we present a protocol for identifying translated smORFs in mammalian systems genome wide. We describe steps for generation of ribosome profiling (Ribo-seq) data, in silico translation of a transcriptome assembly to create an ORF database, and computational analysis of Ribo-seq to score individual smORFs for translation. Identification of translated smORFs is the first step to studying the functions of microproteins. For complete details on the use and execution of this protocol, please refer to Martinez et al.1.

Colonic epithelial repair is a key determinant of health. Repair involves changes in epithelial differentiation, an extensive proliferative response, and upregulation of regeneration-associated fetal-like transcripts, including Ly6a (Sca-1), that represent Yap1 and interferon targets. However, little is known about how this regenerative program terminates and how homeostasis is restored during injury and inflammation. Here we show that, after the initial entry into the regenerative state, the subsequent upregulation of tumor necrosis factor (TNF) receptor 2 (R2, TNFR2, Tnfrsf1b) clears the regenerative signaling and restores homeostatic patterns of epithelial differentiation. Targeted deletion of epithelial TNFR2 in vivo and in colonoid cultures revealed persistent expression of Ly6a, hyperproliferation, and reduced secretory differentiation. Moreover, mice lacking epithelial TNFR2 also failed to complete colon ulcer healing, suggesting that partial resolution of regenerative signaling is essential for the completion of the repair process. These results demonstrate how epithelial cells dynamically leverage a colitis-associated cytokine to choreograph repair.

The heteroplasmic state of eukaryotic cells allows for cryptic accumulation of defective mitochondrial genomes (mtDNA). Purifying selection mechanisms operate to remove such dysfunctional mtDNAs. We found that activators of programmed cell death (PCD), including the CED-3 and CSP-1 caspases, the BH3-only protein CED-13, and PCD corpse engulfment factors, are required in C. elegans to attenuate germline abundance of a 3.1-kb mtDNA deletion mutation, uaDf5, which is normally stably maintained in heteroplasmy with wildtype mtDNA. In contrast, removal of CED-4/Apaf1 or a mutation in the CED-4-interacting prodomain of CED-3, do not increase accumulation of the defective mtDNA, suggesting induction of a non-canonical germline PCD mechanism or non-apoptotic action of the CED-13/caspase axis. We also found that the abundance of germline mtDNAuaDf5 reproducibly increases with age of the mothers. This effect is transmitted to the offspring of mothers, with only partial intergenerational removal of the defective mtDNA. In mutants with elevated mtDNAuaDf5 levels, this removal is enhanced in older mothers, suggesting an age-dependent mechanism of mtDNA quality control. Indeed, we found that both steady-state and age-dependent accumulation rates of uaDf5 are markedly decreased in long-lived, and increased in short-lived, mutants. These findings reveal that regulators of both PCD and the aging program are required for germline mtDNA quality control and its intergenerational transmission.

Here, we present a protocol for isolating human hepatocytes and neural progenitor cells from normal and nonalcoholic steatohepatitis livers. We describe steps for perfusion for scaled-up liver cell isolation and optimization of chemical digestion to achieve maximal yield and cell viability. We then detail a liver cell cryopreservation and potential applications, such as the use of human liver cells as a tool to link experimental and translational research.

Type 2 diabetes (T2D) is associated with compromised identity of insulin-producing pancreatic islet β cells, characterized by inappropriate production of other islet cell-enriched hormones. Here, we examined how hormone misexpression was influenced by the MAFA and MAFB transcription factors, closely related proteins that maintain islet cell function. Mice specifically lacking MafA in β cells demonstrated broad, population-wide changes in hormone gene expression with an overall gene signature closely resembling islet gastrin+ (Gast+) cells generated under conditions of chronic hyperglycemia and obesity. A human β cell line deficient in MAFB, but not one lacking MAFA, also produced a GAST+ gene expression pattern. In addition, GAST was detected in human T2D β cells with low levels of MAFB. Moreover, evidence is provided that human MAFB can directly repress GAST gene transcription. These results support a potentially novel, species-specific role for MafA and MAFB in maintaining adult mouse and human β cell identity, respectively. Here, we discuss the possibility that induction of Gast/GAST and other non-β cell hormones, by reduction in the levels of these transcription factors, represents a dysfunctional β cell signature.

Stentor coeruleus provides a unique opportunity to study how cells regulate nuclear shape because its macronucleus undergoes a rapid, dramatic, and developmentally regulated shape change. We found that the volume of the macronucleus increases during coalescence, suggesting an inflation-based mechanism. When the nuclear transport factor, CSE1, is knocked down by RNAi, the shape and volume changes of the macronucleus are attenuated, and nuclear morphology is altered. CSE1 protein undergoes a dynamic relocalization correlated with nuclear shape changes, being mainly cytoplasmic prior to nuclear coalescence, and accumulating inside the macronucleus during coalescence. At the end of regeneration, CSE1 protein levels are reduced as the macronucleus returns to its pre-coalescence volume. We propose a model in which nuclear transport via CSE1 is required to increase the volume of the macronucleus, thereby decreasing the surface-to-volume ratio and driving coalescence of the nodes into a single mass.

Fusion of multivesicular bodies (MVBs) with the plasma membrane results in the secretion of intraluminal vesicles (ILVs), or exosomes. The sorting of one exosomal cargo RNA, miR223, is facilitated by the RNA-binding protein, YBX1 (Shurtleff et al., 2016). We found that miR223 specifically binds a cold shock domain (CSD) of YBX1 through a 5 proximal sequence motif UCAGU that may represent a binding site or structural feature required for sorting. Prior to sorting into exosomes, most of the cytoplasmic miR223 resides in mitochondria. An RNA-binding protein localized to the mitochondrial matrix, YBAP1, appears to serve as a negative regulator of miR223 enrichment into exosomes. miR223 levels decreased in the mitochondria and increased in exosomes after loss of YBAP1. We observed YBX1 shuttle between mitochondria and endosomes in live cells. YBX1 also partitions into P body granules in the cytoplasm (Liu et al., 2021). We propose a model in which miR223 and likely other miRNAs are stored in mitochondria and are then mobilized by YBX1 to cytoplasmic phase condensate granules for capture into invaginations in the endosome that give rise to exosomes.

Ketone bodies are short-chain fatty acids produced in the liver during periods of limited glucose availability that provide an alternative energy source for the brain, heart, and skeletal muscle. Beyond this metabolic role, β-hydroxybutyrate (BHB), is gaining recognition as a signaling molecule. Lysine β-hydroxybutyrylation (Kbhb) is a newly discovered post-translational modification in which BHB is covalently attached to lysine ε-amino groups. This protein adduct is metabolically sensitive, dependent on BHB concentration, and found on proteins in multiple intracellular compartments. Therefore, Kbhb is hypothesized to be an important component of ketone body-regulated physiology. Kbhb on histones is proposed to be an epigenetic regulator, which links metabolic alterations to gene expression. However, we found that the widely used antibody against β-hydroxybutyrylated lysine 9 on histone H3 (H3K9bhb) also recognizes other modification(s) that likely include acetylation. Therefore, caution must be used when interpreting gene regulation data acquired with the H3K9bhb antibody.

Although budding yeast has been extensively used as a model organism for studying organelle functions and intracellular vesicle trafficking, whether it possesses an independent endocytic early/sorting compartment that sorts endocytic cargos to the endo-lysosomal pathway or the recycling pathway has long been unclear. The structure and properties of the endocytic early/sorting compartment differ significantly between organisms; in plant cells, the trans-Golgi network (TGN) serves this role, whereas in mammalian cells a separate intracellular structure performs this function. The yeast syntaxin homolog Tlg2p, widely localizing to the TGN and endosomal compartments, is presumed to act as a Q-SNARE for endocytic vesicles, but which compartment is the direct target for endocytic vesicles remained unanswered. Here we demonstrate by high-speed and high-resolution 4D imaging of fluorescently labeled endocytic cargos that the Tlg2p-residing compartment within the TGN functions as the early/sorting compartment. After arriving here, endocytic cargos are recycled to the plasma membrane or transported to the yeast Rab5-residing endosomal compartment through the pathway requiring the clathrin adaptors GGAs. Interestingly, Gga2p predominantly localizes at the Tlg2p-residing compartment, and the deletion of GGAs has little effect on another TGN region where Sec7p is present but suppresses dynamics of the Tlg2-residing early/sorting compartment, indicating that the Tlg2p- and Sec7p-residing regions are discrete entities in the mutant. Thus, the Tlg2p-residing region seems to serve as an early/sorting compartment and function independently of the Sec7p-residing region within the TGN.

Flavin adenine dinucleotide (FAD) interacts with flavoproteins to mediate oxidation-reduction reactions required for cellular energy demands. Not surprisingly, mutations that alter FAD binding to flavoproteins cause rare inborn errors of metabolism (IEMs) that disrupt liver function and render fasting intolerance, hepatic steatosis, and lipodystrophy. In our study, depleting FAD pools in mice with a vitamin B2-deficient diet (B2D) caused phenotypes associated with organic acidemias and other IEMs, including reduced body weight, hypoglycemia, and fatty liver disease. Integrated discovery approaches revealed B2D tempered fasting activation of target genes for the nuclear receptor PPARα, including those required for gluconeogenesis. We also found PPARα knockdown in the liver recapitulated B2D effects on glucose excursion and fatty liver disease in mice. Finally, treatment with the PPARα agonist fenofibrate activated the integrated stress response and refilled amino acid substrates to rescue fasting glucose availability and overcome B2D phenotypes. These findings identify metabolic responses to FAD availability and nominate strategies for the management of organic acidemias and other rare IEMs.

The ability to sense and respond to changes in cellular oxygen levels is critical for aerobic organisms and requires a molecular oxygen sensor. The prototypical sensor is the oxygen-dependent enzyme PHD: hypoxia inhibits its ability to hydroxylate the transcription factor HIF, causing HIF to accumulate and trigger the classic HIF-dependent hypoxia response. A small handful of other oxygen sensors are known, all of which are oxygen-dependent enzymes. However, hundreds of oxygen-dependent enzymes exist among aerobic organisms, raising the possibility that additional sensors remain to be discovered. This review summarizes known and potential hypoxia sensors among human O2-dependent enzymes and highlights their possible roles in hypoxia-related adaptation and diseases.

Translocation sequencing can be used to assess mechanisms of DNA repair and identify genome-wide double-strand breaks (DSBs) accessible to DNA repair machinery. Here, we present a protocol for mapping double-strand DNA break sites across the genome with translocation capture sequencing. Bait DSBs are introduced using a Cas9 nuclease and repaired by the host cell, connecting bait DSBs to other DSBs. Repair sites are detected by isolating bait site DNA, cleaving normal sequence to enrich off-site repair, and next-generation sequencing. For complete details on the use and execution of this protocol, please refer to Switonski et al. (2021).1.

Time-lapse microscopy is the only method that can directly capture the dynamics and heterogeneity of fundamental cellular processes at the single-cell level with high temporal resolution. Successful application of single-cell time-lapse microscopy requires automated segmentation and tracking of hundreds of individual cells over several time points. However, segmentation and tracking of single cells remain challenging for the analysis of time-lapse microscopy images, in particular for widely available and non-toxic imaging modalities such as phase-contrast imaging. This work presents a versatile and trainable deep-learning model, termed DeepSea, that allows for both segmentation and tracking of single cells in sequences of phase-contrast live microscopy images with higher precision than existing models. We showcase the application of DeepSea by analyzing cell size regulation in embryonic stem cells.

Here, we present a protocol for electrotaxis of large epithelial cell sheets without compromising the integrity of cell epithelia in a high-throughput customized directed current electrotaxis chamber. We describe the fabrication and use of polydimethylsiloxane stencils to control the size and shape of human keratinocyte cell sheets. We detail cell tracking, cell sheet contour assay, and particle image velocimetry to reveal the spatial and temporal motility dynamics of cell sheets. This approach is applicable to other collective cell migration studies. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2022).1.

Here, we present a protocol to generate a murine model of liver metastasis by directly injecting tumor cells into the portal vein under ultrasound guidance. We describe steps for animal and cell preparation and two techniques for injecting tumor cells. One technique is freehand, while the other technique is device-assisted using a 3D-printed prototype device. Finally, we describe tumor surveillance with bioluminescent imaging.

Many studies worldwide have shown that students and pre-service teachers have misconceptions about cell biology concepts. In most cases, teachers are the source of their students' misconceptions. In this regard, we conducted this study to investigate if Moroccan pre-service teachers of life and earth sciences have a good understanding of cell biology concepts, and to find which concepts they misconceive. We developed and validated a three-tier diagnostic test with 20 items concerning cell biology concepts. The test was based on knowledge related to cell biology included in the Moroccan secondary school life and earth sciences' curriculum, which the pre-service teachers will teach. The use of a three-tier test allowed us to differentiate incorrect answers due to misconceptions from those related to a lack of knowledge and correct answers related to strong knowledge, from lucky guess. This study shows that pre-service teachers share several cell biology misconceptions, and that they misconceive the following concepts: cell, gene, hormone, nerve impulse and immune response. Misconceptions detected may impede pre-service teachers' learning and may be transmitted to their students, which requires development of a training strategy to overcome these obstacles. [ABSTRACT FROM AUTHOR]

Aim– to develop anin vitromethod for assessing the activity of the inflammasome under conditions of hyperuricemic stimulation of inflammatory interleukins. Material and methods.Whole blood cells of donors and patients with hyperuricemia and exacerbation of gouty arthritis diluted with RPMI were culturedin vitroin the presence of different concentrations of uric acid. The production of cytokines in the cell growth media of hematopoietic cells stimulated with uric acid was evaluated using an enzyme-linked immunosorbent assay (ELISA). Results.By simulating the hyperuricemiain vivo, anin vitrocell-based bioassay was developed to stimulate blood cells of individual donors with uric acid. Using the developedin vitroHyperuricemia-hemotest bioassay, quantitative differences were found in the production of inflammatory cytokines by the blood cells of potentially healthy donors and patients with hyperuricemia and gouty arthritis. Conclusion.As a new approach in personalized diagnostics, a hyperuricemic (HU)-hemotest system was developed, which can serve as anin vitrocell model for studying the activation of inflammasome by inflammatory signaling molecules in gouty arthritis.

Abstract Background This study aimed to evaluate dentin wear and biological performance of desensitizing materials. Methods Seventy bovine root dentin blocks were sectioned. Half of the surface of each specimen was untreated (control) and the other half was immersed in EDTA and treated with the following desensitizing materials: placebo varnish (PLA), fluoride varnish (FLU), sodium fluoride (NaF) varnish + sodium trimetaphosphate (TMP), universal adhesive (SBU), S-PRG varnish (SPRG), biosilicate (BIOS), and amelotin solution (AMTN). After application, the specimens were submitted to an erosive-abrasive challenge and the wear analyzed by optical profilometer. Serial dilutions of extracts obtained from the culture medium containing discs impregnated with those desensitizers were applied on fibroblasts and odontoblasts-like cells cultures. Cytotoxicity and production of total protein (TP) by colorimetric assays were determined after 24 h. Data were statistically analyzed using Kruskal-Wallis, Dunn’s, One-way ANOVA and Tukey tests (p ≤ 0.05). Results No dentin wear was observed only for SBU. The lowest dentin wear was observed for AMTN and TMP. Cell viability was significantly reduced after treatment with undiluted extracts of PLA, FLU, TMP and SBU in fibroblasts and TMP and SBU in odontoblast-like cells. SPRG, BIOS and AMTN were cytocompatible at all dilutions tested. Considering TP results, no statistical difference was observed among the groups and high levels for TP were observed after TMP and FLU treatments. Conclusions Universal adhesive system may protect dentin with opened tubules from wear after challenge. Extracts of adhesive and fluoride varnishes presented cytotoxic mainly on fibroblasts. The enamel protein may be a future alternative to treat dentin with opened tubules because it may cause low wear under erosive-abrasive challenge with low cytotoxic effects.

Background: This study aimed to evaluate dentin wear and biological performance of desensitizing materials. Methods: Seventy bovine root dentin blocks were sectioned. Half of the surface of each specimen was untreated (control) and the other half was immersed in EDTA and treated with the following desensitizing materials: placebo varnish (PLA), fluoride varnish (FLU), sodium fluoride (NaF) varnish + sodium trimetaphosphate (TMP), universal adhesive (SBU), S-PRG varnish (SPRG), biosilicate (BIOS), and amelotin solution (AMTN). After application, the specimens were submitted to an erosive-abrasive challenge and the wear analyzed by optical profilometer. Serial dilutions of extracts obtained from the culture medium containing discs impregnated with those desensitizers were applied on fibroblasts and odontoblasts-like cells cultures. Cytotoxicity and production of total protein (TP) by colorimetric assays were determined after 24 h. Data were statistically analyzed using Kruskal-Wallis, Dunn's, One-way ANOVA and Tukey tests (p ≤ 0.05). Results: No dentin wear was observed only for SBU. The lowest dentin wear was observed for AMTN and TMP. Cell viability was significantly reduced after treatment with undiluted extracts of PLA, FLU, TMP and SBU in fibroblasts and TMP and SBU in odontoblast-like cells. SPRG, BIOS and AMTN were cytocompatible at all dilutions tested. Considering TP results, no statistical difference was observed among the groups and high levels for TP were observed after TMP and FLU treatments. Conclusions: Universal adhesive system may protect dentin with opened tubules from wear after challenge. Extracts of adhesive and fluoride varnishes presented cytotoxic mainly on fibroblasts. The enamel protein may be a future alternative to treat dentin with opened tubules because it may cause low wear under erosive-abrasive challenge with low cytotoxic effects. [ABSTRACT FROM AUTHOR]

Robotic arms are now commonplace in diverse settings and are poised to play a crucial role in automating laboratory tasks. However, biological experiments remain challenging for automation due to their dependence on human factors, such as researchers' skills and experience. This article introduces robotic automation and remote control for both general and biological research tasks through a modularized platform comprising a robotic arm, auxiliary tools, and software. This platform facilitates fully automated or remote execution of key experiments in chemistry and biology, including liquid handling, mixing, cell seeding, culturing, and genetic manipulation. The robot interfaces seamlessly with standard laboratory equipment and operates remotely in real time through an online program. Integration of a vision system via robotic arm webcams ensures precise positioning and object localization, enhancing accuracy. This modularized robotic platform signifies a substantial advancement in lab automation, promising enhanced efficiency, reproducibility, and scientific progress compared to human‐led experiments. [ABSTRACT FROM AUTHOR]