Dissecting cell death pathways in fed-batch bioreactors.

Chinese hamster ovary (CHO) cells are widely used for production of biologics including therapeutic monoclonal antibodies. Cell death in CHO cells is a significant factor in biopharmaceutical production, impacting both product yield and quality. Apoptosis has previously been described as the major form of cell death occurring in CHO cells in bioreactors. However, these studies were undertaken when less was known about non-apoptotic cell death pathways. Here, we report the occurrence of non-apoptotic cell death in an industrial antibody-producing CHO cell line during fed-batch culture. Under standard conditions, crucial markers of apoptosis were not observed despite a decrease in viability towards the end of the culture; only by increasing stress within the system did we observe caspase activation indicative of apoptosis. In contrast, markers of parthanatos and ferroptosis were observed during standard fed-batch culture, indicating that these non-apoptotic cell death pathways contribute to viability loss under these conditions. These findings pave the way for targeting non-conventional cell death pathways to improve viability and biologic production in CHO cells.

Phosphorylation of Syntaxin 4 by the Insulin Receptor Drives Exocytic SNARE Complex Formation to Deliver GLUT4 to the Cell Surface.

A major consequence of insulin binding its receptor on fat and muscle cells is the stimulation of glucose transport into these tissues. This is achieved through an increase in the exocytic trafficking rate of the facilitative glucose transporter GLUT4 from intracellular stores to the cell surface. Delivery of GLUT4 to the cell surface requires the formation of functional SNARE complexes containing Syntaxin 4, SNAP23, and VAMP2. Insulin stimulates the formation of these complexes and concomitantly causes phosphorylation of Syntaxin 4. Here, we use a combination of biochemistry and cell biological approaches to provide a mechanistic link between these observations. We present data to support the hypothesis that Tyr-115 and Tyr-251 of Syntaxin 4 are direct substrates of activated insulin receptors, and that these residues modulate the protein's conformation and thus regulate the rate at which Syntaxin 4 forms SNARE complexes that deliver GLUT4 to the cell surface. This report provides molecular details on how the cell regulates SNARE-mediated membrane traffic in response to an external stimulus.

GLUT4 dispersal at the plasma membrane of adipocytes: a super-resolved journey.

In adipose tissue, insulin stimulates glucose uptake by mediating the translocation of GLUT4 from intracellular vesicles to the plasma membrane. In 2010, insulin was revealed to also have a fundamental impact on the spatial distribution of GLUT4 within the plasma membrane, with the existence of two GLUT4 populations at the plasma membrane being defined: (1) as stationary clusters and (2) as diffusible monomers. In this model, in the absence of insulin, plasma membrane-fused GLUT4 are found to behave as clusters. These clusters are thought to arise from exocytic events that retain GLUT4 at their fusion sites; this has been proposed to function as an intermediate hub between GLUT4 exocytosis and re-internalisation. By contrast, insulin stimulation induces the dispersal of GLUT4 clusters into monomers and favours a distinct type of GLUT4-vesicle fusion event, known as fusion-with-release exocytosis. Here, we review how super-resolution microscopy approaches have allowed investigation of the characteristics of plasma membrane-fused GLUT4 and further discuss regulatory step(s) involved in the GLUT4 dispersal machinery, introducing the scaffold protein EFR3 which facilitates localisation of phosphatidylinositol 4-kinase type IIIα (PI4KIIIα) to the cell surface. We consider how dispersal may be linked to the control of transporter activity, consider whether macro-organisation may be a widely used phenomenon to control proteins within the plasma membrane, and speculate on the origin of different forms of GLUT4-vesicle exocytosis.

EHD2 regulates plasma membrane integrity and downstream insulin receptor signaling events.

Adipocyte dysfunction is a crucial driver of insulin resistance and type 2 diabetes. We identified EH domain-containing protein 2 (EHD2) as one of the most highly upregulated genes at the early stage of adipose-tissue expansion. EHD2 is a dynamin-related ATPase influencing several cellular processes, including membrane recycling, caveolae dynamics, and lipid metabolism. Here, we investigated the role of EHD2 in adipocyte insulin signaling and glucose transport. Using C57BL6/N EHD2 knockout mice under short-term high-fat diet conditions and 3T3-L1 adipocytes we demonstrate that EHD2 deficiency is associated with deterioration of insulin signal transduction and impaired insulin-stimulated GLUT4 translocation. Furthermore, we show that lack of EHD2 is linked with altered plasma membrane lipid and protein composition, reduced insulin receptor expression, and diminished insulin-dependent SNARE protein complex formation. In conclusion, these data highlight the importance of EHD2 for the integrity of the plasma membrane milieu, insulin receptor stability, and downstream insulin receptor signaling events, involved in glucose uptake and ultimately underscore its role in insulin resistance and obesity.

Phosphorylation of the N-terminus of Syntaxin-16 controls interaction with mVps45 and GLUT4 trafficking in adipocytes.

The ability of insulin to stimulate glucose transport in muscle and fat cells is mediated by the regulated delivery of intracellular vesicles containing glucose transporter-4 (GLUT4) to the plasma membrane, a process known to be defective in disease such as Type 2 diabetes. In the absence of insulin, GLUT4 is sequestered in tubules and vesicles within the cytosol, collectively known as the GLUT4 storage compartment. A subset of these vesicles, known as the 'insulin responsive vesicles' are selectively delivered to the cell surface in response to insulin. We have previously identified Syntaxin16 (Sx16) and its cognate Sec1/Munc18 protein family member mVps45 as key regulatory proteins involved in the delivery of GLUT4 into insulin responsive vesicles. Here we show that mutation of a key residue within the Sx16 N-terminus involved in mVps45 binding, and the mutation of the Sx16 binding site in mVps45 both perturb GLUT4 sorting, consistent with an important role of the interaction of these two proteins in GLUT4 trafficking. We identify Threonine-7 (T7) as a site of phosphorylation of Sx16 in vitro. Mutation of T7 to D impairs Sx16 binding to mVps45 in vitro and overexpression of T7D significantly impaired insulin-stimulated glucose transport in adipocytes. We show that both AMP-activated protein kinase (AMPK) and its relative SIK2 phosphorylate this site. Our data suggest that Sx16 T7 is a potentially important regulatory site for GLUT4 trafficking in adipocytes.

Solar-Driven CO2 Conversion via Optimized Photothermal Catalysis in a Lotus Pod Structure.

Photothermal CO2 reduction is one of the most promising routes to efficiently utilize solar energy for fuel production at high rates. However, this reaction is currently limited by underdeveloped catalysts with low photothermal conversion efficiency, insufficient exposure of active sites, low active material loading, and high material cost. Herein, we report a potassium-modified carbon-supported cobalt (K+ -Co-C) catalyst mimicking the structure of a lotus pod that addresses these challenges. As a result of the designed lotus-pod structure which features an efficient photothermal C substrate with hierarchical pores, an intimate Co/C interface with covalent bonding, and exposed Co catalytic sites with optimized CO binding strength, the K+ -Co-C catalyst shows a record-high photothermal CO2 hydrogenation rate of 758 mmol gcat -1 h-1 (2871 mmol gCo -1 h-1 ) with a 99.8 % selectivity for CO, three orders of magnitude higher than typical photochemical CO2 reduction reactions. We further demonstrate with this catalyst effective CO2 conversion under natural sunlight one hour before sunset during the winter season, putting forward an important step towards practical solar fuel production.

Synergizing Electron and Heat Flows in Photocatalyst for Direct Conversion of Captured CO2.

We report a ternary hybrid photocatalyst architecture with tailored interfaces that boost the utilization of solar energy for photochemical CO2 reduction by synergizing electron and heat flows in the photocatalyst. The photocatalyst comprises cobalt phthalocyanine (CoPc) molecules assembled on multiwalled carbon nanotubes (CNTs) that are decorated with nearly monodispersed cadmium sulfide quantum dots (CdS QDs). The CdS QDs absorb visible light and generate electron-hole pairs. The CNTs rapidly transfer the photogenerated electrons from CdS to CoPc. The CoPc molecules then selectively reduce CO2 to CO. The interfacial dynamics and catalytic behavior are clearly revealed by time-resolved and in situ vibrational spectroscopies. In addition to serving as electron highways, the black body property of the CNT component can create local photothermal heating to activate amine-captured CO2 , namely carbamates, for direct photochemical conversion without additional energy input.

Anti-racist strategies for clinical and translational research: Design, implementation, and lessons learned from a new course.

Translational research should examine racism and bias and improve health equity. We designed and implemented a course for the Master of Science in Clinical Investigation program of the Northwestern University Clinical and Translational Sciences Institute. We describe curriculum development, content, outcomes, and revisions involving 36 students in 2 years of "Anti-Racist Strategies for Clinical and Translational Science." Ninety-six percent of students reported they would recommend the course. Many reported changes in research approaches based on course content. A course designed to teach anti-racist research design is feasible and has a positive short-term impact on learners.

Efficient electrocatalytic valorization of chlorinated organic water pollutant to ethylene.

Electrochemistry can provide an efficient and sustainable way to treat environmental waters polluted by chlorinated organic compounds. However, the electrochemical valorization of 1,2-dichloroethane (DCA) is currently challenged by the lack of a catalyst that can selectively convert DCA in aqueous solutions into ethylene. Here we report a catalyst comprising cobalt phthalocyanine molecules assembled on multiwalled carbon nanotubes that can electrochemically decompose aqueous DCA with high current and energy efficiencies. Ethylene is produced at high rates with unprecedented ~100% Faradaic efficiency across wide electrode potential and reactant concentration ranges. Kinetic studies and density functional theory calculations reveal that the rate-determining step is the first C-Cl bond breaking, which does not involve protons-a key mechanistic feature that enables cobalt phthalocyanine/carbon nanotube to efficiently catalyse DCA dechlorination and suppress the hydrogen evolution reaction. The nanotubular structure of the catalyst enables us to shape it into a flow-through electrified membrane, which we have used to demonstrate >95% DCA removal from simulated water samples with environmentally relevant DCA and electrolyte concentrations.

Aqueous Photoelectrochemical CO2 Reduction to CO and Methanol over a Silicon Photocathode Functionalized with a Cobalt Phthalocyanine Molecular Catalyst.

We report a precious-metal-free molecular catalyst-based photocathode that is active for aqueous CO2 reduction to CO and methanol. The photoelectrode is composed of cobalt phthalocyanine molecules anchored on graphene oxide which is integrated via a (3-aminopropyl)triethoxysilane linker to p-type silicon protected by a thin film of titanium dioxide. The photocathode reduces CO2 to CO with high selectivity at potentials as mild as 0 V versus the reversible hydrogen electrode (vs RHE). Methanol production is observed at an onset potential of -0.36 V vs RHE, and reaches a peak turnover frequency of 0.18 s-1 . To date, this is the only molecular catalyst-based photoelectrode that is active for the six-electron reduction of CO2 to methanol. This work puts forth a strategy for interfacing molecular catalysts to p-type semiconductors and demonstrates state-of-the-art performance for photoelectrochemical CO2 reduction to CO and methanol.

Health Care Access and Use Among Children & Adolescents Exposed to Parental Incarceration-United States, 2019.

OBJECTIVE: The United States has the highest incarceration rate in the world, with spillover impacts on 5 million children with an incarcerated parent. Children exposed to parental incarceration (PI) have suboptimal health care access, use, and outcomes in adulthood compared to their peers. However, little is known about their access and utilization during childhood. We evaluated relationships between PI and health care use and access throughout childhood and adolescence. METHODS: We analyzed the nationally representative 2019 National Health Interview Survey Child Sample to examine cross-sectional associations between exposure to incarceration of a residential caregiver, access to care, and health care use among children aged 2-17. Respondents were asked about measures of preventive care access, unmet needs due to cost, and acute care use over the last year. We estimated changes associated with PI exposure using multiple logistic regression models adjusted for age, sex, race, ethnicity, parental education, family structure, rurality, income, insurance status, and disability. RESULTS: Of 7405 sample individuals, 467 (weighted 6.2% [95% CI 5.5-6.9]) were exposed to PI. In adjusted analyses to produce national estimates, exposure to PI was associated with an additional 2.2 million children lacking a usual source of care, 2 million with forgone dental care needs, 1.2 million with delayed mental health care needs, and 865,000 with forgone mental health care needs. CONCLUSIONS: Exposure to PI was associated with worse access to a usual source of care and unmet dental and mental health care needs. Our findings highlight the need for early intervention by demonstrating that these barriers emerge during childhood and adolescence.

Pleiotropic effects of Syntaxin16 identified by gene editing in cultured adipocytes.

Adipocytes play multiple roles in the regulation of glucose metabolism which rely on the regulation of membrane traffic. These include secretion of adipokines and serving as an energy store. Central to their energy storing function is the ability to increase glucose uptake in response to insulin, mediated through translocation of the facilitative glucose transporter GLUT4 to the cell surface. The trans-Golgi reticulum localized SNARE protein syntaxin 16 (Sx16) has been identified as a key component of the secretory pathway required for insulin-regulated trafficking of GLUT4. We used CRISPR/Cas9 technology to generate 3T3-L1 adipocytes lacking Sx16 to understand the role of the secretory pathway on adipocyte function. GLUT4 mRNA and protein levels were reduced in Sx16 knockout adipocytes and insulin stimulated GLUT4 translocation to the cell surface was reduced. Strikingly, neither basal nor insulin-stimulated glucose transport were affected. By contrast, GLUT1 levels were upregulated in Sx16 knockout cells. Levels of sortilin and insulin regulated aminopeptidase were also increased in Sx16 knockout adipocytes which may indicate an upregulation of an alternative GLUT4 sorting pathway as a compensatory mechanism for the loss of Sx16. In response to chronic insulin stimulation, Sx16 knockout adipocytes exhibit elevated insulin-independent glucose transport and significant alterations in lactate metabolism. We further show that the adipokine secretory pathways are impaired in Sx16 knockout cells. Together this demonstrates a role for Sx16 in the control of glucose transport, the response to elevated insulin, cellular metabolic profiles and adipocytokine secretion.

GLUT4 translocation and dispersal operate in multiple cell types and are negatively correlated with cell size in adipocytes.

The regulated translocation of the glucose transporter, GLUT4, to the surface of adipocytes and muscle is a key action of insulin. This is underpinned by the delivery and fusion of GLUT4-containing vesicles with the plasma membrane. Recent studies have revealed that a further action of insulin is to mediate the dispersal of GLUT4 molecules away from the site of GLUT4 vesicle fusion with the plasma membrane. Although shown in adipocytes, whether insulin-stimulated dispersal occurs in other cells and/or is exhibited by other proteins remains a matter of debate. Here we show that insulin stimulates GLUT4 dispersal in the plasma membrane of adipocytes, induced pluripotent stem cell-derived cardiomyocytes and HeLa cells, suggesting that this phenomenon is specific to GLUT4 expressed in all cell types. By contrast, insulin-stimulated dispersal of TfR was not observed in HeLa cells, suggesting that the mechanism may be unique to GLUT4. Consistent with dispersal being an important physiological mechanism, we observed that insulin-stimulated GLUT4 dispersal is reduced under conditions of insulin resistance. Adipocytes of different sizes have been shown to exhibit distinct metabolic properties: larger adipocytes exhibit reduced insulin-stimulated glucose transport compared to smaller cells. Here we show that both GLUT4 delivery to the plasma membrane and GLUT4 dispersal are reduced in larger adipocytes, supporting the hypothesis that larger adipocytes are refractory to insulin challenge compared to their smaller counterparts, even within a supposedly homogeneous population of cells.

Electrochemical CO2 Reduction in the Presence of Impurities: Influences and Mitigation Strategies.

The electrochemical conversion of waste CO2 into useful fuels and chemical products is a promising approach to reduce CO2 emissions; however, several challenges still remain to be addressed. Thus far, most CO2 reduction studies use pure CO2 as the gas reactant, but CO2 emissions typically contain a number of gas impurities, such as nitrogen oxides, oxygen gas, and sulfur oxides. Gas impurities in CO2 can pose a significant obstacle for efficient CO2 electrolysis because they can influence the reaction and catalyst. This Minireview highlights early examples of CO2 reduction studies using mixed-gas feeds, explores strategies to sustain CO2 reduction in the presence of gas impurities, and discusses their implications for future progress in this emerging field.

Diabetes is accompanied by changes in the levels of proteins involved in endosomal GLUT4 trafficking in obese human skeletal muscle.

INTRODUCTION: The regulated delivery of the glucose transporter GLUT4 from intracellular stores to the plasma membrane underpins insulin-stimulated glucose transport. Insulin-stimulated glucose transport is impaired in skeletal muscle of patients with type-2 diabetes, and this may arise because of impaired intracellular trafficking of GLUT4. However, molecular details of any such impairment have not been described. We hypothesized that GLUT4 and/or levels of proteins involved in intracellular GLUT4 trafficking may be impaired in skeletal muscle in type-2 diabetes and tested this in obese individuals without and without type-2 diabetes. METHODS: We recruited 12 participants with type-2 diabetes and 12 control participants. All were overweight or obese with BMI of 25-45 kg/m2 . Insulin sensitivity was measured using an insulin suppression test (IST), and vastus lateralis biopsies were taken in the fasted state. Cell extracts were immunoblotted to quantify levels of a range of proteins known to be involved in intracellular GLUT4 trafficking. RESULTS: Obese participants with type-2 diabetes exhibited elevated fasting blood glucose and increased steady state glucose infusion rates in the IST compared with controls. Consistent with this, skeletal muscle from those with type-2 diabetes expressed lower levels of GLUT4 (30%, p = .014). Levels of Syntaxin4, a key protein involved in GLUT4 vesicle fusion with the plasma membrane, were similar between groups. By contrast, we observed reductions in levels of Syntaxin16 (33.7%, p = 0.05), Sortilin (44%, p = .006) and Sorting Nexin-1 (21.5%, p = .039) and -27 (60%, p = .001), key proteins involved in the intracellular sorting of GLUT4, in participants with type-2 diabetes. CONCLUSIONS: We report significant reductions of proteins involved in the endosomal trafficking of GLUT4 in skeletal muscle in obese people with type 2 diabetes compared with age- and weight-matched controls. These abnormalities of intracellular GLUT4 trafficking may contribute to reduced whole body insulin sensitivity.

EFR3 and phosphatidylinositol 4-kinase IIIα regulate insulin-stimulated glucose transport and GLUT4 dispersal in 3T3-L1 adipocytes.

Insulin stimulates glucose transport in muscle and adipocytes. This is achieved by regulated delivery of intracellular glucose transporter (GLUT4)-containing vesicles to the plasma membrane where they dock and fuse, resulting in increased cell surface GLUT4 levels. Recent work identified a potential further regulatory step, in which insulin increases the dispersal of GLUT4 in the plasma membrane away from the sites of vesicle fusion. EFR3 is a scaffold protein that facilitates localization of phosphatidylinositol 4-kinase type IIIα to the cell surface. Here we show that knockdown of EFR3 or phosphatidylinositol 4-kinase type IIIα impairs insulin-stimulated glucose transport in adipocytes. Using direct stochastic reconstruction microscopy, we also show that EFR3 knockdown impairs insulin stimulated GLUT4 dispersal in the plasma membrane. We propose that EFR3 plays a previously unidentified role in controlling insulin-stimulated glucose transport by facilitating dispersal of GLUT4 within the plasma membrane.

TFIIIC-based chromatin insulators through eukaryotic evolution.

Eukaryotic chromosomes are divided into domains with distinct structural and functional properties, such as differing levels of chromatin compaction and gene transcription. Domains of relatively compact chromatin and minimal transcription are termed heterochromatic, whereas euchromatin is more open and actively transcribed. Insulators separate these domains and maintain their distinct features. Disruption of insulators can cause diseases such as cancer. Many insulators contain tRNA genes (tDNAs), examples of which have been shown to block the spread of activating or silencing activities. This characteristic of specific tDNAs is conserved through evolution, such that human tDNAs can serve as barriers to the spread of silencing in fission yeast. Here we demonstrate that tDNAs from the methylotrophic fungus Pichia pastoris can function effectively as insulators in distantly-related budding yeast. Key to the function of tDNAs as insulators is TFIIIC, a transcription factor that is also required for their expression. TFIIIC binds additional loci besides tDNAs, some of which have insulator activity. Although the mechanistic basis of TFIIIC-based insulation has been studied extensively in yeast, it is largely uncharacterized in metazoa. Utilising publicly-available genome-wide ChIP-seq data, we consider the extent to which mechanisms conserved from yeast to man may suffice to allow efficient insulation by TFIIIC in the more challenging chromatin environments of metazoa and suggest features that may have been acquired during evolution to cope with new challenges. We demonstrate the widespread presence at human tDNAs of USF1, a transcription factor with well-established barrier activity in vertebrates. We predict that tDNA-based insulators in higher organisms have evolved through incorporation of modules, such as binding sites for factors like USF1 and CTCF that are absent from yeasts, thereby strengthening function and providing opportunities for regulation between cell types.

Large scale, single-cell FRET-based glucose uptake measurements within heterogeneous populations.

Fluorescent biosensors are powerful tools allowing the concentration of metabolites and small molecules, and other properties such as pH and molecular crowding to be measured inside live single cells. The technology has been hampered by lack of simple software to identify cells and quantify biosensor signals in single cells. We have developed a new software package, FRETzel, to address this gap and demonstrate its use by measuring insulin-stimulated glucose uptake in individual fat cells of varying sizes for the first time. Our results support the long-standing hypothesis that larger fat cells are less sensitive to insulin than smaller ones, a finding that has important implications for the battle against type 2 diabetes. FRETzel has been optimized using the messy and crowded environment of cultured adipocytes, demonstrating its utility for quantification of FRET biosensors in a wide range of other cell types, including fibroblasts and yeast via a simple user-friendly quantitative interface.