Publications

( 0 )
Armbruster, M;Naskar, S;Garcia, JP;Sommer, M;Kim, E;Adam, Y;Haydon, PG;Boyden, ES;Cohen, AE;Dulla, CG.
Nature neuroscience,
25,
(5),
607-616,
(2022)
Astrocytes are glial cells that interact with neuronal synapses via their distal processes, where they remove glutamate and potassium (K+) from the extracellular space following neuronal activity. Astrocyte clearance of both glutamate and K+ is voltage dependent, but astrocyte membrane potential (Vm) is thought to be largely invariant. As a result, these voltage dependencies have not been considered relevant to astrocyte function. Using genetically encoded voltage indicators to enable the measurement of Vm at peripheral astrocyte processes (PAPs) in mice, we report large, rapid, focal and pathway-specific depolarizations in PAPs during neuronal activity. These activity-dependent astrocyte depolarizations are driven by action potential-mediated presynaptic K+ efflux and electrogenic glutamate transporters. We find that PAP depolarization inhibits astrocyte glutamate clearance during neuronal activity, enhancing neuronal activation by glutamate. This represents a novel class of subcellular astrocyte membrane dynamics and a new form of astrocyte-neuron interaction.
Hegazi, S;Cheng, AH;Krupp, JJ;Tasaki, T;Liu, J;Szulc, DA;Ling, HH;Rios Garcia, J;Seecharran, S;Basiri, T;Amiri, M;Anwar, Z;Ahmad, S;Nayal, K;Sonenberg, N;Liu, BH;Cheng, HM;Levine, JD;Cheng, HM.
Nature communications,
13,
(1),
1594,
(2022)
Ubiquitin ligases control the degradation of core clock proteins to govern the speed and resetting properties of the circadian pacemaker. However, few studies have addressed their potential to regulate other cellular events within clock neurons beyond clock protein turnover. Here, we report that the ubiquitin ligase, UBR4/POE, strengthens the central pacemaker by facilitating neuropeptide trafficking in clock neurons and promoting network synchrony. Ubr4-deficient mice are resistant to jetlag, whereas poe knockdown flies are prone to arrhythmicity, behaviors reflective of the reduced axonal trafficking of circadian neuropeptides. At the cellular level, Ubr4 ablation impairs the export of secreted proteins from the Golgi apparatus by reducing the expression of Coronin 7, which is required for budding of Golgi-derived transport vesicles. In summary, UBR4/POE fulfills a conserved and unexpected role in the vesicular trafficking of neuropeptides, a function that has important implications for circadian clock synchrony and circuit-level signal processing.
Maqdasy, S;Lecoutre, S;Renzi, G;Frendo-Cumbo, S;Rizo-Roca, D;Moritz, T;Juvany, M;Hodek, O;Gao, H;Couchet, M;Witting, M;Kerr, A;Bergo, MO;Choudhury, RP;Aouadi, M;Zierath, JR;Krook, A;Mejhert, N;Rydén, M.
Nature metabolism,
4,
(2),
190-202,
(2022)
The mechanisms promoting disturbed white adipocyte function in obesity remain largely unclear. Herein, we integrate white adipose tissue (WAT) metabolomic and transcriptomic data from clinical cohorts and find that the WAT phosphocreatine/creatine ratio is increased and creatine kinase-B expression and activity is decreased in the obese state. In human in vitro and murine in vivo models, we demonstrate that decreased phosphocreatine metabolism in white adipocytes alters adenosine monophosphate-activated protein kinase activity via effects on adenosine triphosphate/adenosine diphosphate levels, independently of WAT beigeing. This disturbance promotes a pro-inflammatory profile characterized, in part, by increased chemokine (C-C motif) ligand 2 (CCL2) production. These data suggest that the phosphocreatine/creatine system links cellular energy shuttling with pro-inflammatory responses in human and murine white adipocytes. Our findings provide unexpected perspectives on the mechanisms driving WAT inflammation in obesity and may present avenues to target adipocyte dysfunction.
Carvelli, A;Setti, A;Desideri, F;Galfrè, SG;Biscarini, S;Santini, T;Colantoni, A;Peruzzi, G;Marzi, MJ;Capauto, D;Di Angelantonio, S;Ballarino, M;Nicassio, F;Laneve, P;Bozzoni, I.
The EMBO journal,
e108918,
(2022)
The transition from dividing progenitors to postmitotic motor neurons (MNs) is orchestrated by a series of events, which are mainly studied at the transcriptional level by analyzing the activity of specific programming transcription factors. Here, we identify a post-transcriptional role of a MN-specific transcriptional unit (MN2) harboring a lncRNA (lncMN2-203) and two miRNAs (miR-325-3p and miR-384-5p) in this transition. Through the use of in vitro mESC differentiation and single-cell sequencing of CRISPR/Cas9 mutants, we demonstrate that lncMN2-203 affects MN differentiation by sponging miR-466i-5p and upregulating its targets, including several factors involved in neuronal differentiation and function. In parallel, miR-325-3p and miR-384-5p, co-transcribed with lncMN2-203, act by repressing proliferation-related factors. These findings indicate the functional relevance of the MN2 locus and exemplify additional layers of specificity regulation in MN differentiation.
Musardo, S;Therin, S;Pelucchi, S;D'Andrea, L;Stringhi, R;Ribeiro, A;Manca, A;Balducci, C;Pagano, J;Sala, C;Verpelli, C;Grieco, V;Edefonti, V;Forloni, G;Gardoni, F;Meli, G;Di Marino, D;Di Luca, M;Marcello, E.
Molecular therapy : the journal of the American Society of Gene Therapy,
(2022)
The development of new therapeutic avenues that target the early stages of Alzheimer’s disease (AD) is urgently necessary. A disintegrin and metalloproteinase domain 10 (ADAM10) is a sheddase that is involved in dendritic spine shaping and limits the generation of amyloid-β. ADAM10 endocytosis increases in the hippocampus of AD patients, resulting in the decreased postsynaptic localization of the enzyme. To restore this altered pathway, we developed a cell-permeable peptide (PEP3) with a strong safety profile that is able to interfere with ADAM10 endocytosis, upregulating the postsynaptic localization and activity of ADAM10. After extensive validation, experiments in a relevant animal model clarified the optimal timing of the treatment window. PEP3 administration was effective for the rescue of cognitive defects in APP/PS1 mice only if administered at an early disease stage. Increased ADAM10 activity promoted synaptic plasticity, as revealed by changes in the molecular compositions of synapses and the spine morphology. Even though further studies are required to evaluate efficacy and safety issues of long-term administration of PEP3, these results provide preclinical evidence to support the therapeutic potential of PEP3 in AD.
Meka, DP;Kobler, O;Hong, S;Friedrich, CM;Wuesthoff, S;Henis, M;Schwanke, B;Krisp, C;Schmuelling, N;Rueter, R;Ruecker, T;Betleja, E;Cheng, T;Mahjoub, MR;Soba, P;Schlüter, H;Fornasiero, EF;Calderon de Anda, F.
Cell reports,
39,
(3),
110686,
(2022)
Microtubule (MT) modifications are critical during axon development, with stable MTs populating the axon. How these modifications are spatially coordinated is unclear. Here, via high-resolution microscopy, we show that early developing neurons have fewer somatic acetylated MTs restricted near the centrosome. At later stages, however, acetylated MTs spread out in soma and concentrate in growing axon. Live imaging in early plated neurons of the MT plus-end protein, EB3, show increased displacement and growth rate near the MTOC, suggesting local differences that might support axon selection. Moreover, F-actin disruption in early developing neurons, which show fewer somatic acetylated MTs, does not induce multiple axons, unlike later stages. Overexpression of centrosomal protein 120 (Cep120), which promotes MT acetylation/stabilization, induces multiple axons, while its knockdown downregulates proteins modulating MT dynamics and stability, hampering axon formation. Collectively, we show how centrosome-dependent MT modifications contribute to axon formation.
Sun, Q;Ye, Y;Gui, A;Sun, X;Xie, S;Zhan, Y;Chen, R;Yan, Y;Gu, J;Qiu, S;Liu, W;Zuo, J;Zhang, Q;Yang, L.
Cancer letters,
537,
215678,
(2022)
Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin’s lymphoma, with the combination of rituximab and chemotherapy being the standard treatment for it. Although rituximab monotherapy has a remarkable response rate, drug resistance with unclear mechanisms and lack of effective second-line therapy limit the survival benefits of patients with lymphoma. Here, we report that MORTALIN is highly expressed and correlates with resistance to rituximab-based therapy and poor survival in patients with DLBCL. Mechanistically, gain- and loss-of-function experiments revealed that the voltage-dependent anion channel 1-binding protein, MORTALIN, regulated Ca2+ release from the endoplasmic reticulum through mitochondria-associated membrane, facilitating AP1-mediated cell proliferation and YY-1-mediated downregulation of FAS in DLBCL cells. These dual mechanisms contribute to rituximab resistance. In mouse models, genetic depletion of MORTALIN markedly increased the antitumor activity of rituximab. We shed mechanistic light on MORTALIN-Ca2+-CaMKII-AP1-mediated proliferation and MORTALIN-Ca2+-CaMKII-inhibited death receptor in DLBCL, leading to rituximab resistance, and propose MORTALIN as a novel target for the treatment of DLBCL.
Hori, T;Eguchi, K;Wang, HY;Miyasaka, T;Guillaud, L;Taoufiq, Z;Mahapatra, S;Yamada, H;Takei, K;Takahashi, T.
eLife,
11,
(2022)
Elevation of soluble wild-type (WT) tau occurs in synaptic compartments in Alzheimer’s disease. We addressed whether tau elevation affects synaptic transmission at the calyx of Held in slices from mice brainstem. Whole-cell loading of WT human tau (h-tau) in presynaptic terminals at 10-20 µM caused microtubule (MT) assembly and activity-dependent rundown of excitatory neurotransmission. Capacitance measurements revealed that the primary target of WT h-tau is vesicle endocytosis. Blocking MT assembly using nocodazole prevented tau-induced impairments of endocytosis and neurotransmission. Immunofluorescence imaging analyses revealed that MT assembly by WT h-tau loading was associated with an increased MT-bound fraction of the endocytic protein dynamin. A synthetic dodecapeptide corresponding to dynamin 1-pleckstrin-homology domain inhibited MT-dynamin interaction and rescued tau-induced impairments of endocytosis and neurotransmission. We conclude that elevation of presynaptic WT tau induces de novo assembly of MTs, thereby sequestering free dynamins. As a result, endocytosis and subsequent vesicle replenishment are impaired, causing activity-dependent rundown of neurotransmission.
Lattmann, E;Deng, T;Walser, M;Widmer, P;Rexha-Lambert, C;Prasad, V;Eichhoff, O;Daube, M;Dummer, R;Levesque, MP;Hajnal, A.
PLoS biology,
20,
(2),
e3001317,
(2022)
Cell invasion is an initiating event during tumor cell metastasis and an essential process during development. A screen of C. elegans orthologs of genes overexpressed in invasive human melanoma cells has identified several components of the conserved DNA pre-replication complex (pre-RC) as positive regulators of anchor cell (AC) invasion. The pre-RC genes function cell-autonomously in the G1-arrested AC to promote invasion, independently of their role in licensing DNA replication origins in proliferating cells. While the helicase activity of the pre-RC is necessary for AC invasion, the downstream acting DNA replication initiation factors are not required. The pre-RC promotes the invasive fate by regulating the expression of extracellular matrix genes and components of the PI3K signaling pathway. Increasing PI3K pathway activity partially suppressed the AC invasion defects caused by pre-RC depletion, suggesting that the PI3K pathway is one critical pre-RC target. We propose that the pre-RC, or a part of it, acts in the postmitotic AC as a transcriptional regulator that facilitates the switch to an invasive phenotype.
Liu, J;Knopp, KA;Rackaityte, E;Wang, CY;Laurie, MT;Sunshine, S;Puschnik, AS;DeRisi, JL.
mBio,
13,
(3),
e0020522,
(2022)
Lymphocytic choriomeningitis virus (LCMV) is a well-studied mammarenavirus that can be fatal in congenital infections. However, our understanding of LCMV and its interactions with human host factors remains incomplete. Here, host determinants affecting LCMV infection were investigated through a genome-wide CRISPR knockout screen in A549 cells, a human lung adenocarcinoma line. We identified and validated a variety of novel host factors that play a functional role in LCMV infection. Among these, knockout of the sialomucin CD164, a heavily glycosylated transmembrane protein, was found to ablate infection with multiple LCMV strains but not other hemorrhagic mammarenaviruses in several cell types. Further characterization revealed a dependency of LCMV entry on the cysteine-rich domain of CD164, including an N-linked glycosylation site at residue 104 in that region. Given the documented role of LCMV with respect to transplacental human infections, CD164 expression was investigated in human placental tissue and placental cell lines. CD164 was found to be highly expressed in the cytotrophoblast cells, an initial contact site for pathogens within the placenta, and LCMV infection in placental cells was effectively blocked using a monoclonal antibody specific to the cysteine-rich domain of CD164. Together, this study identifies novel factors associated with LCMV infection of human tissues and highlights the importance of CD164, a sialomucin that previously had not been associated with viral infection. IMPORTANCE Lymphocytic choriomeningitis virus (LCMV) is a human-pathogenic mammarenavirus that can be fatal in congenital infections. Although frequently used in the study of persistent infections in the field of immunology, aspects of this virus’s life cycle remain incomplete. For example, while viral entry has been shown to depend on a cell adhesion molecule, DAG1, genetic knockout of this gene allows for residual viral infection, implying that additional receptors can mediate cell entry. The significance of our study is the identification of host factors important for successful infection, including the sialomucin CD164, which had not been previously associated with viral infection. We demonstrated that CD164 is essential for LCMV entry into human cells and can serve as a possible therapeutic target for treatment of congenital infection.
Bai, Q;Yang, C;Yang, M;Pei, Z;Zhou, X;Liu, J;Ji, H;Li, G;Wu, M;Qin, Y;Wang, Q;Wu, L.
Analytical chemistry,
94,
(6),
2901-2911,
(2022)
Elucidating the intrinsic relationship between mitochondrial pH (pHm) fluctuation and lipid droplets (LDs) formation is vital in cell physiology. The development of small-molecular fluorescent probes for discrimination and simultaneous visualization of pHm fluctuation toward LDs has not yet been reported. In this work, utilizing pH-driven polarity-reversible hemicyanine and rhodamine derivatives, a multifunctional fluorescent probe is developed for selectively identifying mitochondria and LDs under specific pH values via dual-emission channels. This rapid-response probe, Hcy-Rh, has two distinct chemical structures under acidic and alkaline circumstances. In acidic conditions, Hcy-Rh exhibits good hydrophilicity that can target mitochondria and display an intense red fluorescence. Conversely, the probe becomes lipophilic under weakly alkaline conditions and targets LDs, showing a strong blue emission. In this manner, Hcy-Rh can selectively label mitochondria and LDs, exhibiting red and blue fluorescence, respectively. Moreover, this ratiometric probe is applied to map pHm changes in living cells under the stimulus with FCCP, NAC, and H2O2. The interplay of LD-mitochondria under oleic acid treatment and starvation-induced autophagy has been studied using this probe at different pH values. In a word, Hcy-Rh is a potential candidate for further exploring mitochondria-LD interaction mechanisms under pHm fluctuation. Moreover, the polarity-dependent strategy is valuable for designing other functional biological probes in imaging multiple organelles.
Tamura, T;Torii, S;Kajiwara, K;Anzai, I;Fujioka, Y;Noda, K;Taguwa, S;Morioka, Y;Suzuki, R;Fauzyah, Y;Ono, C;Ohba, Y;Okada, M;Fukuhara, T;Matsuura, Y.
PLoS pathogens,
18,
(6),
e1010593,
(2022)
Flaviviruses, which are globally distributed and cause a spectrum of potentially severe illnesses, pose a major threat to public health. Although Flaviviridae viruses, including flaviviruses, possess similar genome structures, only the flaviviruses encode the non-structural protein NS1, which resides in the endoplasmic reticulum (ER) and is secreted from cells after oligomerization. The ER-resident NS1 is known to be involved in viral genome replication, but the essential roles of secretory NS1 in the virus life cycle are not fully understood. Here we characterized the roles of secretory NS1 in the particle formation of flaviviruses. We first identified an amino acid residue essential for the NS1 secretion but not for viral genome replication by using protein-protein interaction network analyses and mutagenesis scanning. By using the recombinant flaviviruses carrying the identified NS1 mutation, we clarified that the mutant flaviviruses employed viral genome replication. We then constructed a recombinant NS1 with the identified mutation and demonstrated by physicochemical assays that the mutant NS1 was unable to form a proper oligomer or associate with liposomes. Finally, we showed that the functions of NS1 that were lost by the identified mutation could be compensated for by the in trans-expression of Erns of pestiviruses and host exchangeable apolipoproteins, which participate in the infectious particle formation of pestiviruses and hepaciviruses in the family Flaviviridae, respectively. Collectively, our study suggests that secretory NS1 plays a role in the particle formation of flaviviruses through its interaction with the lipid membrane.
Allen, SL;Seabright, AP;Quinlan, JI;Dhaliwal, A;Williams, FR;Fine, NHF;Hodson, DJ;Armstrong, MJ;Elsharkaway, AM;Greig, CA;Lai, YC;Lord, JM;Lavery, GG;Breen, L.
Cells,
11,
(7),
(2022)
Sarcopenia is a common complication affecting liver disease patients, yet the underlying mechanisms remain unclear. We aimed to elucidate the cellular mechanisms that drive sarcopenia progression using an in vitro model of liver disease. C2C12 myotubes were serum and amino acid starved for 1-h and subsequently conditioned with fasted ex vivo serum from four non-cirrhotic non-alcoholic fatty liver disease patients (NAFLD), four decompensated end-stage liver disease patients (ESLD) and four age-matched healthy controls (CON) for 4- or 24-h. After 4-h C2C12 myotubes were treated with an anabolic stimulus (5 mM leucine) for 30-min. Myotube diameter was reduced following treatment with serum from ESLD compared with CON (-45%) and NAFLD (-35%; p < 0.001 for both). A reduction in maximal mitochondrial respiration (24% and 29%, respectively), coupling efficiency (~12%) and mitophagy (~13%) was identified in myotubes conditioned with NAFLD and ESLD serum compared with CON (p < 0.05 for both). Myostatin (43%, p = 0.04) and MuRF-1 (41%, p = 0.03) protein content was elevated in myotubes treated with ESLD serum compared with CON. Here we highlight a novel, experimental platform to further probe changes in circulating markers associated with liver disease that may drive sarcopenia and develop targeted therapeutic interventions.
Vignoli, B;Canossa, M.
Cells,
11,
(9),
(2022)
Neurons release and respond to brain-derived neurotrophic factor (BDNF) with bursts of brain activity. BDNF action is known to extend to peri-synaptic astrocytes, contributing to synaptic strengthening. This implies that astrocytes have a set of dynamic responses, some of which might be secondary to activation of the tropomyosin tyrosine kinase B (TrkB) receptor. Here, we assessed the contribution of BDNF to long-term synaptic potentiation (LTP), by specifically deleting TrkB in cortical astrocytes. TrkB deletion had no effect on LTP induction, stabilization and maintenance, indicating that TrkB signaling in astrocytes is extraneous to transducing BDNF activity for synaptic strengthening.
Yan, Z;Ao, X;Liang, X;Chen, Z;Liu, Y;Wang, P;Wang, D;Liu, Z;Liu, X;Zhu, J;Zhou, S;Zhou, P;Gu, Y.
Respiratory research,
23,
(1),
104,
(2022)
Ionizing radiation (IR) can induce pulmonary fibrosis by causing epithelial mesenchymal transition (EMT), but the exact mechanism has not been elucidated. To investigate the molecular mechanism of how radiation induces pulmonary fibrosis by altering miR-486-3p content and thus inducing EMT.The changes of miR-486-3p in cells after irradiation were detected by RT-qPCR. Western blot was used to detect the changes of cellular epithelial marker protein E-cadherin, mesenchymal marker N-cadherin, Vimentin and other proteins. The target gene of miR-486-3p was predicted by bioinformatics method and the binding site was verified by dual luciferase reporter system. In vivo experiments, adeno-associated virus (AAV) was used to carry miR-486-3p mimic to lung. Radiation-induced pulmonary fibrosis (RIPF) model was constructed by 25Gy60Co γ-rays. The structural changes of mouse lung were observed by HE and Masson staining. The expression of relevant proteins in mice was detected by immunohistochemistry.IR could decrease the miR-486-3p levels in vitro and in vivo, and that effect was closely correlated to the occurrence of RIPF. The expression of Snail, which induces EMT, was shown to be restrained by miR-486-3p. Therefore, knockdown of Snail blocked the EMT process induced by radiation or knockdown of miR-486-3p. In addition, the molecular mechanism underlying the IR-induced miRNA level reduction was explored. The increased in BCL6 could inhibit the formation of pri-miR-486-3p, thereby reducing the levels of miR-486-3p in the alveolar epithelial cells, which would otherwise promote EMT and contribute to RIPF by targeting Snail.IR can exacerbate RIPF in mice by activating the transcription factor BCL6, which inhibits the transcription of miR-486-3p and decreases its content, which in turn increases the content of the target gene slug and triggers EMT.
Goodwin, K;Jaslove, JM;Tao, H;Zhu, M;Hopyan, S;Nelson, CM.
iScience,
25,
(3),
103838,
(2022)
Smooth muscle guides the morphogenesis of several epithelia during organogenesis, including the mammalian airways. However, it remains unclear how airway smooth muscle differentiation is spatiotemporally patterned and whether it originates from transcriptionally distinct mesenchymal progenitors. Using single-cell RNA-sequencing of embryonic mouse lungs, we show that the pulmonary mesenchyme contains a continuum of cell identities, but no transcriptionally distinct progenitors. Transcriptional variability correlates with spatially distinct sub-epithelial and sub-mesothelial mesenchymal compartments that are regulated by Wnt signaling. Live-imaging and tension-sensors reveal compartment-specific migratory behaviors and cortical forces and show that sub-epithelial mesenchyme contributes to airway smooth muscle. Reconstructing differentiation trajectories reveals early activation of cytoskeletal and Wnt signaling genes. Consistently, Wnt activation induces the earliest stages of smooth muscle differentiation and local accumulation of mesenchymal F-actin, which influences epithelial morphology. Our single-cell approach uncovers the principles of pulmonary mesenchymal patterning and identifies a morphogenetically active mesenchymal layer that sculpts the airway epithelium.
Trayford, C;Crosbie, D;Rademakers, T;van Blitterswijk, C;Nuijts, R;Ferrari, S;Habibovic, P;LaPointe, V;Dickman, M;van Rijt, S.
ACS Applied Nano Materials,
5,
(3),
3237-3251,
(2022)
Stem cell (SC)-based therapies hold the potential to revolutionize therapeutics by enhancing the body’s natural repair processes. Currently, there are only three SC therapies with marketing authorization within the European Union. To optimize outcomes, it is important to understand the biodistribution and behavior of transplanted SCs in vivo. A variety of imaging agents have been developed to trace SCs; however, they mostly lack the ability to simultaneously monitor the SC function and biodistribution at high resolutions. Here, we report the synthesis and application of a nanoparticle (NP) construct consisting of a gold NP core coated with rhodamine B isothiocyanate (RITC)-doped mesoporous silica (AuMS). The MS layer further contained a thiol-modified internal surface and an amine-modified external surface for dye conjugation. Highly fluorescent AuMS of three different sizes were successfully synthesized. The NPs were non-toxic and efficiently taken up by limbal epithelial SCs (LESCs). We further showed that we can functionalize AuMS with a reactive oxygen species (ROS)-sensitive fluorescent dye using two methods, loading the probe into the mesopores, with or without additional capping by a lipid bilayer, and by covalent attachment to surface and/or mesoporous-functionalized thiol groups. All four formulations displayed a ROS concentration-dependent increase in fluorescence. Further, in an ex vivo SC transplantation model, a combination of optical coherence tomography and fluorescence microscopy was used to synergistically identify AuMS-labeled LESC distribution at micrometer resolution. Our AuMS constructs allow for multimodal imaging and simultaneous ROS sensing of SCs and represent a promising tool for in vivo SC tracing.
Qiu, W;Baasch, T;Laurell, T.
Physical Review Applied,
17,
(4),
(2022)
A high acoustic energy density is required in the acoustic resonator to increase the throughput of acoustophoresis devices. In this study, through both experiments and numerical simulations, we find that the energy density in bulk-wave-acoustophoresis devices can be enhanced by actuating the device from the side. Based on qualitative free-flow focusing experiments, side actuation shows clear superiority to bottom actuation under various input powers and flow rates. Quantitative measurements using confocal microparticle image velocimetry confirm an increase by a factor of 4 in energy density using side actuation. Numerical simulations reveal that side actuation leads to significant symmetry breaking, which accounts for strong acoustic fields in the channel, and the device energy-conversion efficiency using side actuation is also higher than that using bottom actuation for different device aspect ratios. Submicrometer particle focusing is performed using an acoustophoresis device with side actuation and more than 90% of 500-nm-diameter particles are focused under a total flow rate of 30μlmin−1 at an input power of 235 mW, achieved without using a power amplifier.
Undvall, E;Garofalo, F;Procopio, G;Qiu, W;Lenshof, A;Laurell, T;Baasch, T.
Physical Review Applied,
17,
(3),
(2022)
The clinical utility of microfluidic techniques is often hampered by an unsatisfying sample throughput. Here, the effect of inertial forces on acoustofluidic particle sorting at high sample throughputs is investigated experimentally and theoretically. Polystyrene particles are acoustically prefocused to obtain precise trajectories. At increased flow rates it is observed that the particle stream is displaced towards the channel center, and above specific flow settings the particles spill over into the center outlet. This effect, coined the spillover effect, illustrates the complex interplay of viscous and inertial forces inside the microchannel. The effect is due to increased bending of the separatrices at the inlet and outlets and not due to the wall-lift force. The impact of the spillover effect on the separation of two different-sized particles is subsequently studied. Efficient sorting is done for subcritical splitting ratios and flow rates, but for close to critical settings or beyond, there is a breakdown of the acoustofluidic separation.
Kaldmäe, M;Vosselman, T;Zhong, X;Lama, D;Chen, G;Saluri, M;Kronqvist, N;Siau, JW;Ng, AS;Ghadessy, FJ;Sabatier, P;Vojtesek, B;Sarr, M;Sahin, C;Österlund, N;Ilag, LL;Väänänen, VA;Sedimbi, S;Arsenian-Henriksson M;Zubarev, RA;Nilsson, L;Koeck, PJB;Rising, A;Abelein, A;Fritz, N;Johansson, J;Lane, DP;Landreh, M.
Structure (London, England : 1993),
(2022)
Disordered proteins pose a major challenge to structural biology. A prominent example is the tumor suppressor p53, whose low expression levels and poor conformational stability hamper the development of cancer therapeutics. All these characteristics make it a prime example of “life on the edge of solubility.” Here, we investigate whether these features can be modulated by fusing the protein to a highly soluble spider silk domain (NT∗). The chimeric protein displays highly efficient translation and is fully active in human cancer cells. Biophysical characterization reveals a compact conformation, with the disordered transactivation domain of p53 wrapped around the NT∗ domain. We conclude that interactions with NT∗ help to unblock translation of the proline-rich disordered region of p53. Expression of partially disordered cancer targets is similarly enhanced by NT∗. In summary, we demonstrate that inducing co-translational folding via a molecular “spindle and thread” mechanism unblocks protein translation in vitro.