Through a network of progressively maturing vesicles, the endosomal system connects the cell’s interior with extracellular space. Intriguingly, this network exhibits a bilateral architecture, ...comprised of a relatively immobile perinuclear vesicle “cloud” and a highly dynamic peripheral contingent. How this spatiotemporal organization is achieved and what function(s) it curates is unclear. Here, we reveal the endoplasmic reticulum (ER)-located ubiquitin ligase Ring finger protein 26 (RNF26) as the global architect of the entire endosomal system, including the trans-Golgi network (TGN). To specify perinuclear vesicle coordinates, catalytically competent RNF26 recruits and ubiquitinates the scaffold p62/sequestosome 1 (p62/SQSTM1), in turn attracting ubiquitin-binding domains (UBDs) of various vesicle adaptors. Consequently, RNF26 restrains fast transport of diverse vesicles through a common molecular mechanism operating at the ER membrane, until the deubiquitinating enzyme USP15 opposes RNF26 activity to allow vesicle release into the cell’s periphery. By drawing the endosomal system’s architecture, RNF26 orchestrates endosomal maturation and trafficking of cargoes, including signaling receptors, in space and time.
Display omitted
•The bulk of the endosomal system and TGN clusters in the perinuclear “cloud”•The ER-located E3 ubiquitin ligase RNF26 retains vesicles in the perinuclear cloud•RNF26 employs the ubiquitin scaffold SQSTM1 to capture specific vesicle adaptors•Opposition between RNF26 and DUB USP15 times release of vesicles for fast transport
The endosomal system exhibits a bilateral architecture, comprised of a relatively immobile perinuclear vesicle “cloud” and a highly dynamic peripheral contingent. How this cloud is organized and what purpose it serves is unknown. Here, we reveal its molecular determinants centered around the ER-located ubiquitin ligase RNF26, capable of retaining the entire endosomal system’s repertoire through a common mechanism operating at the ER membrane. Countered by the deubiquitinating enzyme USP15, RNF26 draws the endosomal system’s architecture, thus orchestrating vesicle maturation and cargo trafficking in space and time.
Dilated cardiomyopathy is a major cause of progressive heart failure. Utilization of stem cell therapy offers a potential means of regenerating viable cardiac tissue. However, a major obstacle to ...stem cell therapy is the delivery and survival of implanted stem cells in the ischemic heart. To address this issue, we have developed a biomimetic aligned nanofibrous cardiac patch and characterized the alignment and function of human inducible pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) cultured on this cardiac patch. This hiPSC-CMs seeded patch was compared with hiPSC-CMs cultured on standard flat cell culture plates.
hiPSC-CMs were cultured on; 1) a highly aligned polylactide-co-glycolide (PLGA) nanofiber scaffold (~50 microns thick) and 2) on a standard flat culture plate. Scanning electron microscopy (SEM) was used to determine alignment of PLGA nanofibers and orientation of the cells on the respective surfaces. Analysis of gap junctions (Connexin-43) was performed by confocal imaging in both the groups. Calcium cycling and patch-clamp technique were performed to measure calcium transients and electrical coupling properties of cardiomyocytes.
SEM demonstrated >90% alignment of the nanofibers in the patch which is similar to the extracellular matrix of decellularized rat myocardium. Confocal imaging of the cardiomyocytes demonstrated symmetrical alignment in the same direction on the aligned nanofiber patch in sharp contrast to the random appearance of cardiomyocytes cultured on a tissue culture plate. The hiPSC-CMs cultured on aligned nanofiber cardiac patches showed more efficient calcium cycling compared with cells cultured on standard flat surface culture plates. Quantification of mRNA with qRT-PCR confirmed that these cardiomyocytes expressed α-actinin, troponin-T and connexin-43 in-vitro.
Overall, our results demonstrated changes in morphology and function of human induced pluripotent derived cardiomyocytes cultured in an anisotropic environment created by an aligned nanofiber patch. In this environment, these cells better approximate normal cardiac tissue compared with cells cultured on flat surface and can serve as the basis for bioengineering of an implantable cardiac patch.
Autoimmune responses against posttranslationally modified antigens are a hallmark of several autoimmune diseases. For example, antibodies against citrullinated protein antigens (ACPA) have shown ...their relevance for the prognosis and diagnosis of rheumatoid arthritis (RA), and have been implicated in disease pathogenesis. It is conceivable that other autoantibody systems, recognizing other posttranslationally modified proteins, are also present in RA. Here, we describe the presence of an autoantibody system that discriminates between citrulline- and homocitrulline-containing antigens in the sera of RA-patients. IgG antibodies recognizing carbamylated (homocitrulline-containing) antigens were present in sera of over 45% of RA-patients. Likewise, anticarbamylated protein (anti-CarP) IgA antibodies were observed in 43% of RA-sera. ACPA and anti-CarP antibodies are distinct autoantibodies because, in selected double-positive patients, the anti-CarP antibody binding to carbamylated antigens could be inhibited by carbamylated antigens, but not by control or citrullinated antigens. Similarly, ACPA-binding to citrullinated antigens could only be inhibited by citrullinated antigens. In line with this observation, 16% of ACPA-negative RA-patients, as measured by a standard ACPA assay, harbored IgG anti-CarP antibodies, whereas 30% of these patients tested positive for IgA anti-CarP antibodies. The presence of anti-CarP antibodies was predictive for a more severe disease course in ACPA-negative patients as measured by radiological progression. Taken together, these data show the presence of a unique autoantibody system recognizing carbamylated, but not citrullinated, protein antigens. These antibodies are predictive for a more severe clinical course in ACPA-negative RA-patients, indicating that anti-CarP antibodies are a unique and relevant serological marker for ACPA-negative RA.
Here, we provide a detailed account of novel experiments employing electron-ion coincidence imaging to discriminate chiral molecules. The full three-dimensional angular scattering distribution of ...electrons is measured after photoexcitation with either left or right circular polarized light. The experiment is performed using a simplified photoelectron-photoion coincidence imaging setup employing only a single particle imaging detector. Results are reported applying this technique to enantiomers of the chiral molecule camphor after three-photon ionization by circularly polarized femtosecond laser pulses at 400 nm and 380 nm. The electron-ion coincidence imaging provides the photoelectron spectrum of mass-selected ions that are observed in the time-of-flight mass spectra. The coincident photoelectron spectra of the parent camphor ion and the various fragment ions are the same, so it can be concluded that fragmentation of camphor happens after ionization. We discuss the forward-backward asymmetry in the photoelectron angular distribution which is expressed in Legendre polynomials with moments up to order six. Furthermore, we present a method, similar to one-photon electron circular dichroism, to quantify the strength of the chiral electron asymmetry in a single parameter. The circular dichroism in the photoelectron angular distribution of camphor is measured to be 8% at 400 nm. The electron circular dichroism using femtosecond multiphoton excitation is of opposite sign and about 60% larger than the electron dichroism observed before in near-threshold one-photon ionization with synchrotron excitation. We interpret our multiphoton ionization as being resonant at the two-photon level with the 3s and 3p Rydberg states of camphor. Theoretical calculations are presented that model the photoelectron angular distribution from a prealigned camphor molecule using density functional theory and continuum multiple scattering X alpha photoelectron scattering calculations. Qualitative agreement is observed between the experimental results and the theoretical calculations of the Legendre moments representing the angular distribution for the two enantiomers. The electron-ion coincidence technique using multiphoton ionization opens new directions in table-top analytical mass-spectrometric applications of mixtures of chiral molecules.
The volume phase transition of microgels is one of the most paradigmatic examples of stimuli-responsiveness, enabling a collapse from a highly swollen microgel state into a densely coiled state by an ...external stimulus. Although well characterized in bulk, it remains unclear how the phase transition is affected by the presence of a confining interface. Here, we demonstrate that the temperature-induced volume phase transition of poly(N-isopropylacrylamide) microgels, conventionally considered an intrinsic molecular property of the polymer, is in fact largely suppressed when the microgel is adsorbed to an air/liquid interface. We further observe a hysteresis in the core morphology and interfacial pressure between heating and cooling cycles. Our results, supported by molecular dynamics simulations, reveal that the dangling polymer chains of microgel particles, spread at the interface under the influence of surface tension, do not undergo any volume phase transition. The balance in free energy responsible for the volume phase transition is fundamentally altered by interfacial confinement. These results imply that important technological properties of such systems, including the temperature-induced destabilization of emulsions, do not occur via a decrease in the interfacial coverage of the microgels.
Processes affecting the fate of perfluorinated organics are of increasing concern due to the global dispersal, persistence, and bioaccumulation of these contaminants. The volatile compound N-ethyl ...perfluorooctane sulfonamidoethanol (N-EtFOSE) and its phosphate esters have been used in protective surface coatings. In this report, we describe the fate of N-EtFOSE in aerobic batch assays. These assays were performed using undiluted activated sludge in serum bottles that were sealed to prevent the escape of N-EtFOSE and volatile transformation products. Separate assays were performed with N-EtFOSE and reported transformation products. N-EtFOSE degraded to N-ethyl perfluorooctane sulfonamido acetic acid (N-EtFOSAA) with an observed first-order rate of 0.99 ± 0.08 day−1 and a pseudosecond order rate of 0.26 ± 0.02 L/mg VSS day−1. N-EtFOSAA underwent further transformation at a slower rate (0.093 ± 0.012 day−1) to N-ethylperfluorooctane sulfonamide (N-EtFOSA). N-EtFOSA then transformed to perfluorooctane sulfonamide (FOSA). FOSA transformed to perfluorooctane sulfinate (PFOSI), and PFOSI transformed to perfluorooctane sulfonate (PFOS). Perfluorooctanoic acid (PFOA) was not detected as a transformation product of any compound. Using the measured rate of N-EtFOSE biotransformation and literature values for phase partitioning and mass transfer in aeration basins, we modeled the fate of N-EtFOSE in a typical activated sludge aeration basin open to the atmosphere. The model predicts that 76% of the N-EtFOSE is stripped into the atmosphere, 5% sorbs to waste solids, 13% undergoes transformation to N-EtFOSAA, and 6% is discharged in the wastewater effluent.
Photogranules are dense, spherical agglomerates of cyanobacteria, microalgae and non-phototrophic microorganisms that have considerable advantages in terms of harvesting and nutrient removal rates ...for light driven wastewater treatment processes. This ecosystem is poorly understood in terms of the microbial community structure and the response of the community to changing abiotic conditions. To get a better understanding, we investigated the effect of hydraulic retention time (HRT) on photogranule formation and community assembly over a period of 148 days. Three laboratory bioreactors were inoculated with field samples from various locations in the Netherlands and operated in sequencing batch mode. The bioreactors were operated at four different HRTs (2.00, 1.00, 0.67, 0.33 days), while retaining the same solid retention time of 7 days. A microbial community with excellent settling characteristics (95–99% separation efficiency) was established within 2–5 weeks. The observed nutrient uptake rates ranged from 24 to 90 mgN L−1 day−1 and from 3.1 to 5.4 mgP L−1 day−1 depending on the applied HRT. The transition from single-cell suspension culture to floccular agglomeration to granular sludge was monitored by microscopy and 16S/18S sequencing. In particular, two important variables for driving aggregation and granulation, and for the structural integrity of photogranules were identified: 1. Extracellular polymeric substances (EPS) with high protein to polysaccharide ratio and 2. specific microorganisms. The key players were found to be the cyanobacteria Limnothrix and Cephalothrix, the colony forming photosynthetic eukaryotes within Chlamydomonadaceae, and the biofilm producing bacteria Zoogloea and Thauera. Knowing the makeup of the microbial community and the operational conditions influencing granulation and bioreactor function is crucial for successful operation of photogranular systems.
Display omitted
•Lower HRT provides a ‘shortcut’ to well settling biomass and photogranules.•Lower HRT increases overall volumetric removal rates.•Correlation network analysis revealed key players in photogranule assembly and function.•Motile filamentous cyanobacteria and EPS producer are important in photogranulation•Nitrification/denitrification is an important removal pathway in photogranules.
Dense active matter is gaining widespread interest due to its remarkable similarity with conventional glass-forming materials. However, active matter is inherently out of equilibrium and even simple ...models such as active Brownian particles (ABPs) and active Ornstein-Uhlenbeck particles (AOUPs) behave markedly differently from their passive counterparts. Controversially, this difference has been shown to manifest itself via either a speedup, slowdown, or nonmonotonic change of the glassy relaxation dynamics. Here we rationalize these seemingly contrasting views on the departure from equilibrium by identifying the ratio of the short-time length scale to the cage length, i.e., the length scale of local particle caging, as a vital and unifying control parameter for active glassy matter. In particular, we explore the glassy dynamics of both thermal and athermal ABPs and AOUPs upon increasing the persistence time. We find that for all studied systems there is an optimum of the dynamics; this optimum occurs when the cage length coincides with the corresponding short-time length scale of the system, which is either the persistence length for athermal systems or a combination of the persistence length and a diffusive length scale for thermal systems. This new insight, for which we also provide a simple physical argument, allows us to reconcile and explain the manifestly disparate departures from equilibrium reported in many previous studies of dense active materials.
PDF
