Publications

( 0 )
Geiser, P;Di Martino, ML;Samperio Ventayol, P;Eriksson, J;Sima, E;Al-Saffar, AK;Ahl, D;Phillipson, M;Webb, DL;Sundbom, M;Hellström, PM;Sellin, ME.
mBio,
12
(1)
(2021)
(6.8)
Enterobacterial pathogens infect the gut by a multistep process, resulting in colonization of both the lumen and the mucosal epithelium. Due to experimental constraints, it remains challenging to address how luminal and epithelium-lodged pathogen populations cross-feed each other in vivo Enteroids are cultured three-dimensional miniature intestinal organs with a single layer of primary intestinal epithelial cells (IECs) surrounding a central lumen. They offer new opportunities to study enterobacterial infection under near-physiological conditions, at a temporal and spatial resolution not attainable in animal models, but remain poorly explored in this context. We employed microinjection, time-lapse microscopy, bacterial genetics, and barcoded consortium infections to describe the complete infection cycle of Salmonella enterica serovar Typhimurium in both human and murine enteroids. Flagellar motility and type III secretion system 1 (TTSS-1) promoted Salmonella Typhimurium targeting of the intraepithelial compartment and breaching of the epithelial barrier. Strikingly, however, TTSS-1 also potently boosted colonization of the enteroid lumen. By tracing the infection over time, we identified a cycle(s) of TTSS-1-driven IEC invasion, intraepithelial replication, and reemergence through infected IEC expulsion as a key mechanism for Salmonella Typhimurium luminal colonization. These findings suggest a positive feed-forward loop, through which IEC invasion by planktonic bacteria fuels further luminal population expansion, thereby ensuring efficient colonization of both the intraepithelial and luminal niches.IMPORTANCE Pathogenic gut bacteria are common causes of intestinal disease. Enteroids-cultured three-dimensional replicas of the mammalian gut-offer an emerging model system to study disease mechanisms under conditions that recapitulate key features of the intestinal tract. In this study, we describe the full life cycle of the prototype gut pathogen Salmonella enterica serovar Typhimurium within human and mouse enteroids. We map the consecutive steps and define the bacterial virulence factors that drive colonization of luminal and epithelial compartments, as well as breaching of the epithelial barrier. Strikingly, our work reveals how bacterial colonization of the epithelium potently fuels expansion also in the luminal compartment, through a mechanism involving the death and expulsion of bacterium-infected epithelial cells. These findings have repercussions for our understanding of the Salmonella infection cycle. Moreover, our work provides a comprehensive foundation for the use of microinjected enteroids to model gut bacterial diseases.
Baruffaldi, D;Pirri, C;Frascella, F.
Bioprinting,
22
, e00135
(2021)
Traditional in vitro culture models are unable to fully reflect the organ microenvironment, due to differences in terms of cell morphology, protein expression, cell-cell and cell-matrix interactions, and drug response. In contrast, the flexibility of bioprinting modes allows for the deposition of cell-containing biomaterials in any free-form-inspired 3D structures on chip. The main purpose of this study was to design and optimize commercially available Carbopol-based 3D printing formulations, because of their many advantages, such as low-cost, the ability to produce clear and stable gels, and the water thickening. For this purpose, three different Carbopol gels (EDT 2020 NF, Ultrez 10 NF and NF-980) were tested in terms of printability and biocompatibility, with lung cancer epithelial (A549) and normal lung fibroblast (MRC-5) cells. This study demonstrates that Carbopol is a promising candidate for the 3D printing of cell-laden constructs, both in terms of rheology and printing performance.
Danylchuk, DI;Jouard, PH;Klymchenko, AS.
Journal of the American Chemical Society,
143
(2)
, 912-924
(2021)
(14.6)
Biomembranes constitute a basis for all compartments of live cells, and therefore, the monitoring of their lipid organization is essential for understanding cell status and activity. However, the sensing and imaging of lipid organization specifically in different organelles of live cells remain challenging. Here, we designed an array of solvatochromic probes based on Nile Red bearing ligands for specific targeting of the endoplasmic reticulum, mitochondria, lysosomes, Golgi apparatus, plasma membranes, and lipid droplets. These polarity-sensitive probes detected variations in the lipid order by changing their emission maximum, as evidenced by fluorescence spectroscopy in model membranes. In colocalization microscopy experiments with reference organelle markers, they exhibited good organelle selectivity. Using two-color fluorescence microscopy, the new probes enabled imaging of the local polarity of organelles in live cells. To exclude the biased effect of the probe design on the sensitivity to the membrane properties, we calibrated all probes in model membranes under the microscope, which enabled the first quantitative description of the lipid order in each organelle of interest. Cholesterol extraction/enrichment confirmed the capacity of the probes to sense the lipid order, revealing that organelles poor in cholesterol are particularly affected by its enrichment. The probes also revealed that oxidative and mechanical stresses produced changes in the local polarity and lipid order that were characteristic for each organelle, with mitochondria and lysosomes being particularly stress sensitive. The new probes constitute a powerful toolbox for monitoring the response of the cells to physical and chemical stimuli at the level of membranes of individual organelles, which remains an underexplored direction in cellular research.
Fattinger, SA;Geiser, P;Samperio Ventayol, P;Di Martino, ML;Furter, M;Felmy, B;Bakkeren, E;Hausmann, A;Barthel-Scherrer, M;Gül, E;Hardt, WD;Sellin, ME.
Mucosal immunology,
(2021)
(6.7)
The gut epithelium is a critical protective barrier. Its NAIP/NLRC4 inflammasome senses infection by Gram-negative bacteria, including Salmonella Typhimurium (S.Tm) and promotes expulsion of infected enterocytes. During the first ~12-24 h, this reduces mucosal S.Tm loads at the price of moderate enteropathy. It remained unknown how this NAIP/NLRC4-dependent tradeoff would develop during subsequent infection stages. In NAIP/NLRC4-deficient mice, S.Tm elicited severe enteropathy within 72 h, characterized by elevated mucosal TNF (>20 pg/mg) production from bone marrow-derived cells, reduced regeneration, excessive enterocyte loss, and a collapse of the epithelial barrier. TNF-depleting antibodies prevented this destructive pathology. In hosts proficient for epithelial NAIP/NLRC4, a heterogeneous enterocyte death response with both apoptotic and pyroptotic features kept S.Tm loads persistently in check, thereby preventing this dire outcome altogether. Our results demonstrate that immediate and selective removal of infected enterocytes, by locally acting epithelium-autonomous NAIP/NLRC4, is required to avoid a TNF-driven inflammatory hyper-reaction that otherwise destroys the epithelial barrier.
Baek, M;Choe, YJ;Bannwarth, S;Kim, J;Maitra, S;Dorn, GW;Taylor, JP;Paquis-Flucklinger, V;Kim, NC.
Nature communications,
12
(1)
, 1924
(2021)
(12.1)
Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) can cause amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). However, the underlying mechanisms are unclear. Here, we generate CHCH10S59L-mutant Drosophila melanogaster and HeLa cell lines to model CHCHD10-associated ALS-FTD. The CHCHD10S59L mutation results in cell toxicity in several tissues and mitochondrial defects. CHCHD10S59L independently affects the TDP-43 and PINK1 pathways. CHCHD10S59L expression increases TDP-43 insolubility and mitochondrial translocation. Blocking TDP-43 mitochondrial translocation with a peptide inhibitor reduced CHCHD10S59L-mediated toxicity. While genetic and pharmacological modulation of PINK1 expression and activity of its substrates rescues and mitigates the CHCHD10S59L-induced phenotypes and mitochondrial defects, respectively, in both Drosophila and HeLa cells. Our findings suggest that CHCHD10S59L-induced TDP-43 mitochondrial translocation and chronic activation of PINK1-mediated pathways result in dominant toxicity, providing a mechanistic insight into the CHCHD10 mutations associated with ALS-FTD.
Spena, S;Cordiglieri, C;Garagiola, I;Peyvandi, F.
International journal of molecular sciences,
22
(4)
(2021)
(4.6)
Hemophilia is an X-linked recessive bleeding disorder. In pregnant women carrier of hemophilia, the fetal sex can be determined by non-invasive analysis of fetal DNA circulating in the maternal blood. However, in case of a male fetus, conventional invasive procedures are required for the diagnosis of hemophilia. Fetal cells, circulating in the maternal bloodstream, are an ideal target for a safe non-invasive prenatal diagnosis. Nevertheless, the small number of cells and the lack of specific fetal markers have been the most limiting factors for their isolation. We aimed to develop monoclonal antibodies (mAbs) against the ribosomal protein RPS4Y1 expressed in male cells. By Western blotting, immunoprecipitation and immunofluorescence analyses performed on cell lysates from male human hepatoma (HepG2) and female human embryonic kidney (HEK293) we developed and characterized a specific monoclonal antibody against the native form of the male RPS4Y1 protein that can distinguish male from female cells. The availability of the RPS4Y1-targeting monoclonal antibody should facilitate the development of novel methods for the reliable isolation of male fetal cells from the maternal blood and their future use for non-invasive prenatal diagnosis of X-linked inherited disease such as hemophilia.
Nasteska, D;Fine, NHF;Ashford, FB;Cuozzo, F;Viloria, K;Smith, G;Dahir, A;Dawson, PWJ;Lai, YC;Bastidas-Ponce, A;Bakhti, M;Rutter, GA;Fiancette, R;Nano, R;Piemonti, L;Lickert, H;Zhou, Q;Akerman, I;Hodson, DJ.
Nature communications,
12
(1)
, 674
(2021)
(12.1)
Transcriptionally mature and immature β-cells co-exist within the adult islet. How such diversity contributes to insulin release remains poorly understood. Here we show that subtle differences in β-cell maturity, defined using PDX1 and MAFA expression, contribute to islet operation. Functional mapping of rodent and human islets containing proportionally more PDX1HIGH and MAFAHIGH β-cells reveals defects in metabolism, ionic fluxes and insulin secretion. At the transcriptomic level, the presence of increased numbers of PDX1HIGH and MAFAHIGH β-cells leads to dysregulation of gene pathways involved in metabolic processes. Using a chemogenetic disruption strategy, differences in PDX1 and MAFA expression are shown to depend on islet Ca2+ signaling patterns. During metabolic stress, islet function can be restored by redressing the balance between PDX1 and MAFA levels across the β-cell population. Thus, preserving heterogeneity in PDX1 and MAFA expression, and more widely in β-cell maturity, might be important for the maintenance of islet function.
Ibayashi, M;Aizawa, R;Tsukamoto, S.
Biochemical and Biophysical Research Communications,
555
, 128-133
(2021)
(3.0)
mRNA decapping is a critical step in posttranscriptional regulation of gene expression in eukaryotes. Although Dcp1a is a well characterized and widely conserved mRNA decapping factor, little is known about its physiological function. To extend our understanding of Dcp1a function in vivo, we employed a transgenic rescue strategy to produce Dcp1a-deficient mice using the CRISPR/Cas9 system. This approach arrowed us to generate heterozygous Dcp1a mice and define the phenotype of Dcp1a-deficient embryos. We found that expression of Dcp1a protein, which is detectable in most mouse tissues, was developmentally regulated through embryonic growth, and that depletion of the Dcp1a gene resulted in embryonic lethality around embryonic day 10.5 (E10.5) concomitant with massive growth retardation and cardiac developmental defects. Moreover, the embryonic lethality was fully rescued by transgenic expression of exogenous human Dcp1a. Together, our results suggest that Dcp1a is required for embryonic growth.
Zhang, J;Shen, W;Collings, C;Vander Meulen, K;Fox, B;Vázquez Ramos, L;Dumesic, J;Ding, S.
Chemical Engineering Journal,
412
, 128660
(2021)
(10.7)
Thermochemical pretreatment is one of the key steps to process lignocellulosic biomass for production of biofuels and biomaterials. γ-valerolactone (GVL) has been used as a green and renewable solvent to efficiently dissolve lignin under acidic conditions and enhance subsequent enzyme digestibility. Further improvement is still required to lower the pretreatment temperature in order to reduce sugar degradation and irreversible lignin condensation, as well as the capital cost. In this study, we compared the use of HCl and H2SO4 as catalysts during GVL pretreatment, and found the performance of GVL-HCl at 100 °C was comparable to that of GVL-H2SO4 at 120 °C in terms of xylan and lignin removal and enzyme digestibility. We further monitored the lignin removal and cellulose accessibility in the cell walls at different pretreatment time points by imaging the changes in lignin auto-fluorescence and in CtCBM3-GFP binding with confocal laser scanning microscopy (CLSM), respectively. We found GVL-HCl pretreatment at a relative low temperature (100 °C) could rapidly remove lignin in the compound middle lamella and cell corner areas, which concurred with the increase of CtCBM3-GFP binding in these areas. Real-time imaging of cell wall degradation by cellulases further revealed that the secondary cell walls could be digested from both cell lumen and CML sides, and eventually fully deconstructed within 24 h. Our results provide new insight into the effects of chloride anions on plant cell wall structure during GVL pretreatment, and offer potential routes to further optimize and enhance the efficiency of GVL-based pretreatment.
Vignoli, B;Sansevero, G;Sasi, M;Rimondini-Giorgini, R;Blum, R;Santi, S;Berardi, N;Lu, B;Canossa, M.
Research Square,
(2021)
Memory consolidation requires astrocytic microdomains for protein recycling; but whether this lays a mechanistic foundation for long-term information storage remains enigmatic. Here we demonstrate that persistent synaptic strengthening invited astrocytic microdomains to convert initially internalized (pro)-brain-derived neurotrophic factor (proBDNF) into active prodomain (BDNFpro) and mature BDNF (mBDNF) for synaptic re-use. While mBDNF activates TrkB, we uncovered a previously unsuspected function for the cleaved BDNFpro, which increases TrkB/SorCS2 receptor complex at post-synaptic sites. Astrocytic BDNFpro release reinforced TrkB phosphorylation to sustain long-term synaptic potentiation and to retain memory in the novel object recognition behavioral test. Thus, the switch from one inactive state to a multi-functional one of the proBDNF provides post-synaptic changes that survive the initial activation (molecular memory). This molecular asset confines local information storage in astrocytic microdomains to selectively support memory circuits.
Burigotto, M;Mattivi, A;Migliorati, D;Magnani, G;Valentini, C;Roccuzzo, M;Offterdinger, M;Pizzato, M;Schmidt, A;Villunger, A;Maffini, S;Fava, LL.
The EMBO journal,
40
(4)
, e104844
(2021)
(9.9)
Centrosome amplification results into genetic instability and predisposes cells to neoplastic transformation. Supernumerary centrosomes trigger p53 stabilization dependent on the PIDDosome (a multiprotein complex composed by PIDD1, RAIDD and Caspase-2), whose activation results in cleavage of p53’s key inhibitor, MDM2. Here, we demonstrate that PIDD1 is recruited to mature centrosomes by the centriolar distal appendage protein ANKRD26. PIDDosome-dependent Caspase-2 activation requires not only PIDD1 centrosomal localization, but also its autoproteolysis. Following cytokinesis failure, supernumerary centrosomes form clusters, which appear to be necessary for PIDDosome activation. In addition, in the context of DNA damage, activation of the complex results from a p53-dependent elevation of PIDD1 levels independently of centrosome amplification. We propose that PIDDosome activation can in both cases be promoted by an ANKRD26-dependent local increase in PIDD1 concentration close to the centrosome. Collectively, these findings provide a paradigm for how centrosomes can contribute to cell fate determination by igniting a signalling cascade.
Birke, R;Ast, J;Roosen, D;Mathes, B;Roßmann, K;Huhn, C;Jones, B;Lehmann, M;Haucke, V;Hodson, D;Broichhagen, J.
bioRxiv,
(2021)
Sulfonated rhodamines that endow xanthene dyes with cellular impermeability are presented. We fuse charged sulfonates to red and far-red dyes to obtain Sulfo549 and Sulfo646, respectively, and further link these to SNAP- and Halo-tag substrates for protein self-labelling. Cellular impermeability is validated in live cell imaging experiments in transfected HEK cells and neurons derived from induced pluripotent stem cells (iPSCs). Lastly, we show that Sulfo646 is amenable to STED nanoscopy by recording membranes of SNAP/Halo-surface-labelled human iPSC-derived neuronal axons. We therefore provide an avenue for rendering dyes impermeable for exclusive extracellular visualization via self-labelling protein tags.
Lim, Y;Berry, B;Viteri, S;McCall, M;Park, EC;Rongo, C;Brookes, PS;Nehrke, K.
Autophagy,
1-13
(2021)
(9.8)
Mitochondrial quality control (MQC) balances organelle adaptation and elimination, and mechanistic crosstalk between the underlying molecular processes affects subsequent stress outcomes. FUNDC1 (FUN14 domain containing 1) is a mammalian mitophagy receptor that responds to hypoxia-reoxygenation (HR) stress. Here, we provide evidence that FNDC-1 is the C. elegans ortholog of FUNDC1, and that its loss protects against injury in a worm model of HR. This protection depends upon ATFS-1, a transcription factor that is central to the mitochondrial unfolded protein response (UPRmt). Global mRNA and metabolite profiling suggest that atfs-1-dependent stress responses and metabolic remodeling occur in response to the loss of fndc-1. These data support a role for FNDC-1 in non-hypoxic MQC, and further suggest that these changes are prophylactic in relation to subsequent HR. Our results highlight functional coordination between mitochondrial adaptation and elimination that organizes stress responses and metabolic rewiring to protect against HR injury.
Graham, K;Spruston, N;Bloss, E.
bioRxiv,
(2021)
Neural circuits within the frontal cortex support the flexible selection of goal-directed behaviors by integrating input from brain regions associated with sensory, emotional, episodic, and semantic memory functions. From a connectomics perspective, determining how these disparate afferent inputs target their synapses to specific cell types in the frontal cortex may prove crucial in understanding circuit-level information processing. Here, we used monosynaptic retrograde rabies mapping to examine the distribution of afferent neurons targeting four distinct classes of local inhibitory interneurons and four distinct classes of excitatory projection neurons in mouse infralimbic cortex. Interneurons expressing parvalbumin, somatostatin, or vasoactive intestinal peptide received a large proportion of inputs from hippocampal regions, while interneurons expressing neuron-derived neurotrophic factor received a large proportion of inputs from thalamic regions. A more moderate hippocampal-thalamic dichotomy was found among the inputs targeting excitatory neurons that project to the basolateral amygdala, lateral entorhinal cortex, nucleus reuniens of the thalamus, and the periaqueductal gray. Together, these results show a prominent bias among hippocampal and thalamic afferent systems in their targeting to genetically or anatomically defined sets of frontal cortical neurons. Moreover, they suggest the presence of two distinct local microcircuits that control how different inputs govern frontal cortical information processing.
Dobramysl, U;Jarsch, IK;Inoue, Y;Shimo, H;Richier, B;Gadsby, JR;Mason, J;Szałapak, A;Ioannou, PS;Correia, GP;Walrant, A;Butler, R;Hannezo, E;Simons, BD;Gallop, JL.
The Journal of cell biology,
220
(4)
(2021)
(8.8)
Assemblies of actin and its regulators underlie the dynamic morphology of all eukaryotic cells. To understand how actin regulatory proteins work together to generate actin-rich structures such as filopodia, we analyzed the localization of diverse actin regulators within filopodia in Drosophila embryos and in a complementary in vitro system of filopodia-like structures (FLSs). We found that the composition of the regulatory protein complex where actin is incorporated (the filopodial tip complex) is remarkably heterogeneous both in vivo and in vitro. Our data reveal that different pairs of proteins correlate with each other and with actin bundle length, suggesting the presence of functional subcomplexes. This is consistent with a theoretical framework where three or more redundant subcomplexes join the tip complex stochastically, with any two being sufficient to drive filopodia formation. We provide an explanation for the observed heterogeneity and suggest that a mechanism based on multiple components allows stereotypical filopodial dynamics to arise from diverse upstream signaling pathways.
Pickford, P;Lucey, M;Rujan, R;McGlone, E;Bitsi, S;Ashford, F;Corrêa, I;Hodson, D;Tomas, A;Deganutti, G;Reynolds, C;Owen, B;Tan, T;Minnion, J;Jones, B;Bloom, S.
bioRxiv,
(2021)
Objective: Glucagon-like peptide-1 and glucagon receptor (GLP-1R/GCGR) co-agonism can maximise weight loss and improve glycaemic control in type 2 diabetes and obesity. In this study we investigated the cellular and metabolic effects of modulating the balance between G protein activation and β-arrestin-2 recruitment at GLP-1R and GCGR using oxyntomodulin (OXM)-derived co-agonists. This strategy has been previously shown to improve the duration of action of GLP-1R mono-agonists by reducing target desensitisation and downregulation. Methods: Dipeptidyl dipeptidase-4 (DPP-4)-resistant OXM analogues were generated and assessed for a variety of cellular readouts. Molecular dynamic simulations were used to gain insights into the molecular interactions involved. In vivo studies were performed in mice to identify effects on glucose homeostasis and weight loss. Results: Ligand-specific reductions in β-arrestin-2 recruitment led to reduced GLP-1R internalisation and prolonged glucose-lowering action in vivo. The putative benefits of GCGR agonism were retained, with equivalent weight loss compared to the GLP-1R mono-agonist liraglutide in spite of a lesser degree of food intake suppression. The compounds tested showed only a minor degree of biased agonism between G protein and β-arrestin-2 recruitment at both receptors and were best classified as partial agonists for the two pathways measured. Conclusions: Diminishing β-arrestin-2 recruitment may be an effective way to increase the therapeutic efficacy of GLP-1R/GCGR co-agonists. These benefits can be achieved by partial rather than biased agonism.
Dindas, J;Dreyer, I;Huang, S;Hedrich, R;Roelfsema, MRG.
The New phytologist,
(2021)
(8.5)
Cytosolic calcium signals are evoked by a large variety of biotic and abiotic stimuli and play an important role in cellular and long distance signalling in plants. While the function of the plasma membrane in cytosolic Ca2+ signalling has been intensively studied, the role of the vacuolar membrane remains elusive. A newly developed vacuolar voltage clamp technique was used in combination with live-cell imaging, to study the role of the vacuolar membrane in Ca2+ and pH homeostasis of bulging root hair cells of Arabidopsis. Depolarisation of the vacuolar membrane caused a rapid increase in the Ca2+ concentration and alkalised the cytosol, while hyperpolarisation led to the opposite responses. The relationship between the vacuolar membrane potential, the cytosolic pH and Ca2+ concentration suggests that a vacuolar H+ /Ca2+ exchange mechanism plays a central role in cytosolic Ca2+ homeostasis. Mathematical modelling further suggests that the voltage-dependent vacuolar Ca2+ homeostat could contribute to calcium signalling when coupled to a recently discovered K+ channel-dependent module for electrical excitability of the vacuolar membrane.
Basilico, B;Ferrucci, L;Ratano, P;Golia, M;Grimaldi, A;Rosito, M;Ferretti, V;Reverte, I;Marrone, M;Giubettini, M;De Turris, V;Salerno, D;Garofalo, S;St-Pierre, M;Carrier, M;Renzi, M;Pagani, F;Raspa, M;Scavizzi, F;Gross, C;Marinelli, S;Tremblay, M;Caprioli, D;Maggi, L;Limatola, C;Di Angelantonio, S;Ragozzino, D.
bioRxiv,
(2021)
Microglial cells are active players in regulating synaptic development and plasticity in the brain. However, how these cells influence the normal functioning of synapses is largely unknown. In this study, we characterized the effects of pharmacological depletion of microglia, achieved by administration of PLX5622, on hippocampal CA3-CA1 synapses of adult wild type mice. Following microglial depletion, we observed a reduction of spontaneous and evoked glutamatergic activity associated with a decrease of dendritic spine density. We also observed the appearance of immature synaptic features accompanied by higher levels of plasticity. In addition, microglia depleted mice showed a deficit in the acquisition of the Novel Object Recognition task. Remarkably, microglial repopulation after PLX5622 withdrawal was associated with the recovery of hippocampal synapses and learning functions. Altogether, these data demonstrate that microglia contribute to normal synaptic functioning in the adult brain and that their removal induces reversible changes in synaptic organization and activity of glutamatergic synapses.
Cai, X;Hu, CH;Wang, J;Zeng, XH;Luo, JX;Li, M;Liu, ZQ;Zheng, YG.
Bioresource technology,
326
, 124768
(2021)
(7.5)
Corncob is an abundant and renewable resource that could be enzymatically hydrolyzed to fermentable sugar. A major impediment in corncob utilization is the low hydrolysis efficiency at high-solids content. This study attempted different pretreatment methods and fed-batch modes to achieve a 25% solids content hydrolysis with high yields. Natural corncobs were compared with acid-treated and acid-alkali-treated corncobs in terms of kinetics parameters, conversion rate and glucose titer. By feeding in batches, a “low amount and high frequency” mode (10%-3%-3%-3%-3%-3%, every 5 h) was confirmed to be optimal for a 25% high-solids hydrolysis system with a cellulase loading of 12 mg/g (7.3 FPU/g), resulted with an 84.4% glucose yield at 96 h. Our results demonstrated that combination of both optimized pretreatment method and fed-batch mode were a favored process model for high-solids hydrolysis of lignocellulose, boosting cellulose hydrolysis efficiency and sugar yields on an industrial scale.
Janská, L;Anandi, L;Kirchberger, N;Marinkovic, Z;Schachtner, L;Guzelsoy, G;Carmona-Fontaine, C.
bioRxiv,
(2021)
There is an urgent need for accurate, scalable, and cost-efficient models of the complexity and heterogeneity of the tumor microenvironment. Here, we detail how to fabricate and use the Metabolic Microenvironment Chamber (MEMIC) – a 3D-printed ex vivo model of intratumoral heterogeneity. A major driver of the cellular and molecular diversity in tumors is the accessibility to the blood stream that provides key resources such as oxygen and nutrients. While some tumor cells have direct access to these resources, many others must survive under progressively more ischemic environments as they reside further from the vasculature. The MEMIC is designed to simulate the differential access to nutrients and allows co-culturing different cell types, such as tumor and immune cells. This system is optimized for live imaging and other microscopy-based approaches and it is a powerful tool to study tumor features such as the effect of nutrient scarcity on tumor-stroma interactions. Due to its adaptable design and full experimental control, the MEMIC can provide novel insights into the tumor microenvironment that would be difficult to obtain via other methods. As a proof of principle, we show that cells can sense gradual changes in metabolite concentration, and tune intracellular cell signaling to form multicellular spatial patterns of cell proliferation. We also show that ischemic macrophages reduce epithelial features in neighboring tumor cells highlighting the power of this system to study cell-cell interactions and non-cell autonomous effects of the metabolic microenvironment. We propose that the MEMIC can be easily adapted to study early development, ischemic stroke, and other systems where multiple cell types interact within heterogeneous environments.