Cell Biology Research Articles

12328 Multimodal Imaging Reveals Changes In Beta Cell Metabolism And Heterogeneity Throughout Pregnancy

Abstract Disclosure: J.M. Cuala: None. T. Singh: None. C. Deng: None. K. Jayapalan: None. S. Dhawan: None. K. White: None. S. Georgia: None. During pregnancy, a unique, transient metabolic stressor, pancreatic islets adapt by balancing hormone production to maintain tight glucose homeostasis. Maternal insulin demand increases as systemic insulin resistance increases. To keep up with the increase in demand, beta cell functional dynamics change throughout pregnancy, including increasing insulin synthesis and lowering the threshold for glucose-stimulated insulin secretion (GSIS). Failure to adapt can lead to gestational diabetes (GD), a complication in 14% of pregnancies worldwide, feeding into a vicious cycle of impaired function and, eventually, hyperglycemia. Fundamentally, beta cell function is dictated by its metabolism, calcium signaling, and insulin content upon glucose stimulation. To better understand how beta cell maladaptation during pregnancy leads to gestational diabetes, we propose to apply a multidisciplinary approach to integrate novel tools to assess function and metabolism fluctuations throughout normal pregnancy. Using live isolated islets from wild-type C57BL/6 (BL6) and InsCre-gCAMP6f fl/fl (INS-gCAMP) mice, we will measure GSIS and take 4D images using metabolic fluorescence lifetime imaging (FLIM) and confocal microscopy to visualize and quantify metabolic and functional changes at different time points at cellular resolution. We expect functional and metabolic capacity levels to increase during the onset of beta cell remodeling (∼Day 8) and continue to peak throughout pregnancy. In contrast, changes in beta cell heterogeneity become more evident at the peaks of beta cell proliferation and expansion (∼Day 11-14). The proposed study will implement novel techniques to establish beta cell functional and metabolic information within a 3D pancreatic islet context throughout different time points of pregnancy. With this, we will provide greater insight into beta cell biology and its adaptation to metabolic stress to better understand the progression of gestational diabetes, which can then be applied to investigate other cellular adaptive dynamics that affect health and disease. Presentation: 6/2/2024

Assessing and interpreting diastolic function in animal models of heart disease

Increasing interest in identifying the causes of and treatments for heart failure with preserved ejection fraction and cardiac fibrosis has spawned a focus on measures of cardiac diastolic function. The methods, their underlying principals and mechanics, and caveats to their measurement were largely worked out decades ago, but some of this seems a bit forgotten as scientists working in the field now have backgrounds more in molecular and cellular biology. This perspective was spawned by seeing the growing number of studies where diastolic function analysis is a key parameter used to justify a given pre-clinical model or to show the consequences of a particular genetic or pharmacological therapy. The goals are to discuss what comprises and influences diastolic function, how it is measured, what the parameters mean and what their limitations are, and what comprises evidence for pathophysiologically meaningful diastolic dysfunction.

Identifying metabolic limitations in the tumor microenvironment.

Solid tumors are characterized by dysfunctional vasculature that limits perfusion and delivery of nutrients to the tumor microenvironment. Limited perfusion coupled with the high metabolic demand of growing tumors has led to the hypothesis that many tumors experience metabolic stress driven by limited availability of nutrients such as glucose, oxygen, and amino acids in the tumor. Such metabolic stress has important implications for the biology of cells in the microenvironment, affecting both disease progression and response to therapies. Recently, techniques have been developed to identify limiting nutrients and resulting metabolic stresses in solid tumors. These techniques have greatly expanded our understanding of the metabolic limitations in tumors. This review will discuss these experimental tools and the emerging picture of metabolic limitations in tumors arising from recent studies using these approaches.

What are the essential determinants of human papillomavirus carcinogenesis?

Human papillomavirus (HPV) infection is the leading viral cause of cancer. Over the past several decades, research on HPVs has provided remarkable insight into human cell biology and into the pathology of viral and non-viral cancers. The HPV E6 and E7 proteins engage host cellular proteins to establish an environment in infected cells that is conducive to virus replication. They rewire host cell signaling pathways to promote proliferation, inhibit differentiation, and limit cell death. The activity of the "high-risk" HPV E6 and E7 proteins is so potent that their dysregulated expression is sufficient to drive the initiation and maintenance of HPV-associated cancers. Consequently, intensive research efforts have aimed to identify the host cell targets of E6 and E7, in part with the idea that some or all of the virus-host interactions would be essential cancer drivers. These efforts have identified a large number of potential binding partners of each oncoprotein. However, over the same time period, parallel research has revealed that a relatively small number of genetic mutations drive carcinogenesis in most non-viral cancers. We therefore propose that a high-priority goal is to identify which of the many targets of E6 and E7 are critical drivers of HPV carcinogenesis. By identifying the cancer-driving targets of E6 and E7, it should be possible to better understand the distinct roles of other targets, perhaps in the viral life cycle, and to focus efforts to develop anti-cancer therapies on the subset of virus-host interactions for which therapeutic intervention would have the greatest impact.

The Buds of Oscarella lobularis (Porifera, Homoscleromorpha): A New Convenient Model for Sponge Cell and Evolutionary Developmental Biology.

The comparative study of the four non-bilaterian phyla (Cnidaria, Placozoa, Ctenophora, and Porifera) provides insights into the origin of bilaterian traits. To complete our knowledge of the cell biology and development of these animals, additional non-bilaterian models are needed. Given the developmental, histological, ecological, and genomic differences between the four sponge classes (Demospongiae, Calcarea, Homoscleromorpha, and Hexactinellida), we have been developing the Oscarella lobularis (Porifera, class Homoscleromorpha) model over the past 15 years. Here, we report a new step forward by inducing, producing, and maintaining in vitro thousands of clonal buds that now make possible various downstream applications. This study provides a full description of bud morphology, physiology, cells and tissues, from their formation to their development into juveniles, using adapted cell staining protocols. In addition, we show that buds have outstanding capabilities of regeneration after being injured and of re-epithelization after complete cell dissociation. Altogether, Oscarella buds constitute a relevant all-in-one sponge model to access a large set of biological processes, including somatic morphogenesis, epithelial morphogenesis, cell fate, body axes formation, nutrition, contraction, ciliary beating, and respiration.

Detection of host cell microprotein impurities in antibody drug products

Chinese hamster ovary (CHO) cells are used to produce almost 90% of therapeutic monoclonal antibodies (mAbs) and antibody fusion proteins (Fc-fusion). The annotation of non-canonical translation events in these cellular factories remains incomplete, limiting our ability to study CHO cell biology and detect host cell protein (HCP) impurities in the final antibody drug product. We utilised ribosome footprint profiling (Ribo-seq) to identify novel open reading frames (ORFs) including N-terminal extensions and thousands of short ORFs (sORFs) predicted to encode microproteins. Mass spectrometry-based HCP analysis of eight commercial antibody drug products (7 mAbs and 1 Fc-fusion protein) using the extended protein sequence database revealed the presence of microprotein impurities. We present evidence that microprotein abundance varies with growth phase and can be affected by the cell culture environment. In addition, our work provides a vital resource to facilitate future studies of non-canonical translation and the regulation of protein synthesis in CHO cell lines.

Pan-cancer analysis of the prognostic and immunological roles of SHP-1/ptpn6

SHP-1, a nonreceptor protein tyrosine phosphatase encoded by ptpn6, has been regarded as a regulatory protein of hematopoietic cell biology for years. However, there is now increasing evidence to support its role in tumors. Thus, the role of ptpn6 for prognosis and immune regulation across 33 tumors was investigated, aiming to explore its functional heterogeneity and clinical significance in pan-cancer. Differential expression of ptpn6 was found between cancer and adjacent normal tissues, and its expression was significantly correlated with the prognosis of tumor patients. In most cancers, ptpn6 expression was significantly associated with immune infiltration. This was further confirmed by ptpn6-related genes/proteins enrichment analysis. Additionally, genetic alterations in ptpn6 was observed in most cancers. As for epigenetic changes, it’s phosphorylation levels significantly altered in 6 tumors, while methylation levels significantly altered in 12 tumors. Notably, the methylation levels of ptpn6 were significantly decreased in 11 tumors, accompanied by its increased expression in 8 of them, suggesting that the hypomethylation may be related to its increased expression. Our results show that ptpn6 plays a specific role in tumor immunity and exerts a pleiotropic effect in a variety of tumors. It can serve as a prognostic factor for some cancers. Especially in LGG, KIRC, UCS and TGCT, the increased expression of ptpn6 is associated with poor prognosis and high immune infiltration. This aids in understanding the role of ptpn6 in tumor biology, and can provide insight into presenting a potential biomarker for poor prognosis and immune infiltration in cancers.

Anesthetic Techniques and Cancer Outcomes: What Is the Current Evidence?

It is almost 2 decades since it was first hypothesized that anesthesia technique might modulate cancer biology and thus potentially influence patients’ long-term outcomes after cancer surgery. Since then, research efforts have been directed towards elucidating the potential pharmacological and physiological basis for the effects of anesthetic and perioperative interventions on cancer cell biology. In this review, we summarize current laboratory and clinical data. Taken together, preclinical studies suggest some biologic plausibility that cancer cell function could be influenced. However, available clinical evidence suggests a neutral effect. Observational studies examining cancer outcomes after surgery of curative intent for many cancer types under a variety of anesthetic techniques have reported conflicting results, but warranting prospective randomized clinical trials (RCTs). Given the large patient numbers and long follow-up times required for adequate power, relatively few such RCTs have been completed to date. With the sole exception of peritumoral lidocaine infiltration in breast cancer surgery, these RCTs have indicated a neutral effect of anesthetic technique on long-term oncologic outcomes. Therefore, unless there are significant new findings from a few ongoing trials, future investigation of how perioperative agents interact with tumor genes that influence metastatic potential may be justified. In addition, building multidisciplinary collaboration to optimize perioperative care of cancer patients will be important.

Glucose Transporter 1 Inhibitors Induce Autophagy and Synergize With Lenvatinib in Thyroid Cancer Cells.

Less differentiated thyroid cancer may upregulate the expression of glucose transporter 1 (GLUT1) and increase glycolytic activity. However, it is uncertain whether GLUT1 can be used as a target for therapy. Thyroid cancer cell lines were treated with two different GLUT1 inhibitors, STF-31 and BAY-876. Functional assays were conducted to evaluate the effects of these inhibitors on cell biology. GLUT1 inhibitors dose-dependently decreased cell growth and clonogenicity of thyroid cancer cells. Cell cycle analysis showed that these inhibitors caused G2/M arrest instead of apoptosis. Additionally, treatment with GLUT1 inhibitors led to the activation of autophagy. In both the Transwell and spheroid models, GLUT1 inhibitors significantly suppressed cell invasiveness. Moreover, GLUT1 inhibitors demonstrated synergistic interactions when combined with lenvatinib. Treatment with GLUT1 inhibitors activates autophagy and provokes cell cycle arrest, accompanied by a decrease in colony formation and invasive capacity in thyroid cancer cells.

A unique cell division protein critical for the assembly of the bacterial divisome.

Identification of unique essential bacterial genes is important for not only the understanding of their cell biology but also the development of new antimicrobials. Here, we report a previously unrecognized core component of the Acinetobacter baumannii divisome. Our results reveal that the protein, termed Aeg1 interacts with multiple cell division proteins, including FtsN, which is required for components of the divisome to localize to the midcell. We demonstrate that the FtsAE202K and FtsBE65A mutants effectively bypassed the need of Aeg1 by A. baumannii, as did the activation variants FtsWM254I and FtsWS274G. Our results suggest that Aeg1 is a cell division protein that arrives at the division site to initiate cell division by recruiting FtsN, which activates FtsQLB and FtsA to induce the septal peptidoglycan synthase FtsWI. The discovery of the new essential cell division protein has provided a new target for the development of antibacterial agents.

Roles of ZEB1 and ZEB2 in E-cadherin expression and cell aggressiveness in head and neck cancer.

Zinc finger E-box binding homeobox 1 (ZEB1) has been identified as a key factor in cancer cell differentiation and metastasis, and has been well studied in the field of cancer cell biology. ZEB2 has a highly similar conformation to ZEB1, but its role in head and neck squamous cell carcinoma (HNSCC) cells is not fully understood. Here, we separately overexpressed ZEB1 and ZEB2 in C57BL/6 mouse oral cancer (MOC) cells and investigated their cellular characteristics, including E-cadherin levels, motile properties, chemoresistance, and metastatic ability in immunocompetent mice. Both ZEB1 and ZEB2 overexpression reduced epithelial traits and converted cells to an aggressive phenotype. Surprisingly, ZEB1 overexpression increased the endogenous level of ZEB2 in MOC cells, and vice versa. The molecular mechanisms underlying these findings remain unclear. However, the in vitro anchorage-independent growth of MOC cells overexpressing ZEB2 was considerably greater than that of MOC cells overexpressing ZEB1. These findings suggest that ZEB2, like ZEB1, has the ability to induce the differentiation of cancer cells into those with highly aggressive traits.

Radial glia progenitor polarity in health and disease.

Radial glia (RG) are the main progenitor cell type in the developing cortex. These cells are highly polarized, with a long basal process spanning the entire thickness of the cortex and acting as a support for neuronal migration. The RG cell terminates by an endfoot that contacts the pial (basal) surface. A shorter apical process also terminates with an endfoot that faces the ventricle, with a primary cilium protruding in the cerebrospinal fluid. These cell domains have particular subcellular compositions that are critical for the correct functioning of RG. When altered, this can affect proper development of the cortex, ultimately leading to cortical malformations, associated with different pathological outcomes. In this review, we focus on the current knowledge concerning the cell biology of these bipolar stem cells and discuss the role of their polarity in health and disease.

Thymic Mimetic Cells: Ontogeny as Immunology.

Medullary thymic epithelial cells (mTECs) generate immunological self-tolerance by ectopically expressing peripheral-tissue antigens (PTAs) within the thymus to preview the peripheral self to maturing Tcells. Recent work, drawing inspiration from old histological observations, has shown that subtypes of mTECs, collectively termed mimetic cells, co-opt developmental programs from throughout the organism to express biologically coherent groups of PTAs. Here, we review key aspects of mimetic cells, especially as they relate to the larger contexts of molecular, cellular, developmental, and evolutionary biology. We highlight lineage-defining transcription factors as key regulators of mimetic cells and speculate as to what other factors, including Aire and the chromatin potential of mTECs, permit mimetic cell differentiation and function. Last, we consider what mimetic cells can teach us about not only the thymus but also other tissues.

Targeting CREB-binding protein (CBP) abrogates colorectal cancer stemness through epigenetic regulation of C-MYC.

Colorectal cancer (CRC) is a common cancer worldwide with an increasing annual incidence. Cancer stem cells (CSCs) play important roles in the occurrence, development, recurrence, and metastasis of CRC. The molecular mechanism regulating the development of colorectal CSCs remains unclear. The discovery of human induced pluripotent stem cells (hiPSCs) through somatic cell reprogramming has revolutionized the fields of stem cell biology and translational medicine. In the present study, we converted hiPSCs into cancer stem-like cells by culture with conditioned medium (CM) from CRC cells. These transformed cells, termed hiPSC-CSCs, displayed cancer stem-like properties, including a spheroid morphology and the expression of both pluripotency and CSC markers. HiPSC-CSCs showed tumorigenic and metastatic abilities in mouse models. The epithelial-mesenchymal transition phenotype was observed in hiPSC-CSCs, which promoted their migration and angiogenesis. Interestingly, upregulation of C-MYC was observed during the differentiation of hiPSC-CSCs. Mechanistically, CREB binding protein (CBP) bound to the C-MYC promoter, while histone deacetylase 1 and 3 (HDAC1/3) dissociated from the promoter, ultimately leading to an increase in histone acetylation and C-MYC transcriptional activation during the differentiation of hiPSC-CSCs. Pharmacological treatment with a CBP inhibitor or abrogation of CBP expression with a CRISPR/Cas9-based strategy reduced the stemness of hiPSC-CSCs. This study demonstrates for the first time that colorectal CSCs can be generated from hiPSCs. The upregulation of C-MYC via histone acetylation plays a crucial role during the conversion process. Inhibition of CBP is a potential strategy for attenuating the stemness of colorectal CSCs.

Development of a novel Japanese eel myoblast cell line for application in cultured meat production

The present study investigates the isolation, analysis, and characterization of primary cultured cells derived from the muscle tissue of Japanese eel (Anguilla japonica), culminating in establishing a spontaneously immortalized myoblast cell line, JEM1129. We isolated satellite cells from eel muscle tissue to establish a foundation for cultured eel meat production. While initial cell cultures contained myoblasts, continued passaging led to a decline in myoblast characteristics and an increase in fibroblast-like cells. RNA-Seq and RT-qPCR analyses showed significant downregulation of well-established markers for satellite cells and myoblasts, such as pax7a and myoD, over successive passages, highlighting a loss of myoblastic traits. Single-cell cloning was employed to overcome this challenge and maintain myoblast purity, leading to the successful creation of the JEM1129 cell line. These JEM1129 cells demonstrated enhanced expression of myoblast marker genes, exceeding the initial primary culture cell population. The cells showed strong myotube formation, particularly when cultured in a differentiation medium, indicating their robust potential for muscle development. The JEM1129 cell line represents a significant advancement in the cultivation of eel muscle cells, offering a promising avenue for cultured meat production. The findings contribute to a deeper understanding of muscle cell biology and provide valuable insights into using fish-derived myoblasts for cultured meat production.

EnhancerNet: a predictive model of cell identity dynamics through enhancer selection.

Understanding how cell identity is encoded by the genome and acquired during differentiation is a central challenge in cell biology. I have developed a theoretical framework called EnhancerNet, which models the regulation of cell identity through the lens of transcription factor-enhancer interactions. I demonstrate that autoregulation in these interactions imposes a constraint on the model, resulting in simplified dynamics that can be parameterized from observed cell identities. Despite its simplicity, EnhancerNet recapitulates a broad range of experimental observations on cell identity dynamics, including enhancer selection, cell fate induction, hierarchical differentiation through multipotent progenitor states and direct reprogramming by transcription factor overexpression. The model makes specific quantitative predictions, reproducing known reprogramming recipes and the complex haematopoietic differentiation hierarchy without fitting unobserved parameters. EnhancerNet provides insights into how new cell types could evolve and highlights the functional importance of distal regulatory elements with dynamic chromatin in multicellular evolution.

Towards a Systemic Concept of the Brain Ishemia Stroke: Monte Carlo Driven in silico Model.

The work proposes a new mathematical model of dynamic processes of a typical spatially heterogeneous biological system, and sets and solves a mathematical problem of modeling the dynamics of the system of neurovascular units of the brain in conditions of ischemic stroke. There is a description of only a small number of mathematical models of stroke in the literature. This model is being studied and a numerical and software implementation of the corresponding mathematical problem is proposed. This work is the first attempt ever aiming to employ a Monte Carlo computational approach for In Silico simulation of the most critical parameters in molecular and cellular pathogenesis of the brain ischemic stroke. In this work, a new mathematical model of the development of ischemic stroke is proposed in the form of a discrete model based on neurovascular units (NVU) as elements. As a result of testing the program with the assignment of empirically selected coefficients, data were obtained on the evolution of the states of the lattice of the cellular automaton of the model for the spread of stroke in a region of the brain tissue. A resulting new theoretical model of the particular pathologically altered biosystem might be taken as a promising tool for further studies in neurology; general pathology and cell biology. For the first time, a mathematical model has been constructed that allows us to represent the spatial dynamics of the development of the affected area in ischemic stroke of the brain, taking into account neurovascular units as single morphofunctional structures.

DNA polymerase ζ has robust reverse transcriptase activity relative to other cellular DNA polymerases.

Cell biology and genetic studies have demonstrated that DNA double strand break (DSB) repair can be performed using an RNA transcript that spans the site of the DNA break as a template for repair. This type of DSB repair requires a reverse transcriptase to convert an RNA sequence into DNA to facilitate repair of the break, rather than copying from a DNA template as in canonical DSB repair. Translesion synthesis (TLS) DNA polymerases (Pol) are often more promiscuous than DNA Pols, raising the notion that reverse transcription could be performed by a TLS Pol. Indeed, several studies have demonstrated that human Pol η has reverse transcriptase activity, while others have suggested that the yeast TLS Pol ζ is involved. Here, we purify all seven known nuclear DNA Pols of Saccharomyces cerevisiae and compare their reverse transcriptase activities. The comparison shows that Pol ζ far surpasses Pol η and all other DNA Pols in reverse transcriptase activity. We find that Pol ζ reverse transcriptase activity is not affected by RPA or RFC/PCNA and acts distributively to make DNA complementary to an RNA template strand. Consistent with prior S. cerevisiae studies performed in vivo, we propose that Pol ζ is the major DNA Pol that functions in the RNA templated DSB repair pathway.

A proteomic atlas of glypican‐3 interacting partners: Identification of alpha‐fetoprotein and other extracellular proteins as potential immunotherapy targets in liver cancer

AbstractAntibody and cell‐based therapeutics targeting cell surface receptors have emerged as a major class of immune therapeutics for treating cancer. However, the number of cell surface targets for cancer immunotherapy remains limited. Glypican‐3 (GPC3) is a cell surface proteoglycan and an oncofetal antigen. In this study, we report a large‐scale tumor‐associated GPC3 co‐immunoprecipitation‐proteomic study using liver cancer xenograft tumors in mice. We identified 153 GPC3‐associated proteins through mass spectrometry. To identify potential drug targets, we categorized GPC3‐associated proteins based on their subcellular locations using UniProt annotations, with a focus on extracellular proteins. Additionally, we annotated differentially expressed proteins in hepatocellular carcinoma versus nontumor liver samples based on the literature, analyzed expression levels in tumor versus normal tissues using TCGA and GTEx databases via GEPIA, and identified prognostic liver cancer markers according to GEPIA. Among GPC3‐associated proteins, immunoglobulin superfamily member 1, alpha‐fetoprotein (AFP), FAT atypical cadherin 1, formin 1, and guanylate cyclase 2 C, were identified as potential therapeutic targets. Furthermore, we validated the direct protein interaction between GPC3 and AFP through immunoprecipitation with purified proteins and through co‐localization imaging using immunofluorescence microscopy. This study provides large proteomic datasets related to GPC3‐associated proteins, enhancing our understanding of glypican biology in cancer cells and offering a new approach to identifying immunotherapy targets.

Nuclear deformability facilitates apical nuclear migration in the developing zebrafish retina.

Nuclear positioning is a crucial aspect of cell and developmental biology. One example is the apical movement of nuclei in neuroepithelia before mitosis, which is essential for proper tissue formation. While the cytoskeletal mechanisms that drive nuclei to the apical side have been explored, the influence of nuclear properties on apical nuclear migration is less understood. Nuclear properties, such as deformability, can be linked to lamin A/C expression levels, as shown in various invitro studies. Interestingly, many nuclei in early development, including neuroepithelial nuclei, express only low levels of lamin A/C. Therefore, we investigated whether increased lamin A expression in the densely packed zebrafish retinal neuroepithelium affects nuclear deformability and, consequently, migration phenomena. We found that overexpressing lamin A in retinal nuclei increases nuclear stiffness, which in turn indeed impairs apical nuclear migration. Interestingly, nuclei that do not overexpress lamin A but are embedded in a stiffer lamin A-overexpressing environment also exhibit impaired apical nuclear migration, indicating that these effects can be cell non-autonomous. Additionally, in the less crowded hindbrain neuroepithelium, only minor effects on apical nuclear migration are observed. Together, this suggests that the material properties of the nucleus influence nuclear movements in a tissue-dependent manner.