Specialized astrocytes mediate glutamatergic gliotransmission in the CNS.

Multimodal astrocyte-neuron communications govern brain circuitry assembly and function1. For example, through rapid glutamate release, astrocytes can control excitability, plasticity and synchronous activity2,3 of synaptic networks, while also contributing to their dysregulation in neuropsychiatric conditions4-7. For astrocytes to communicate through fast focal glutamate release, they should possess an apparatus for Ca2+-dependent exocytosis similar to neurons8-10. However, the existence of this mechanism has been questioned11-13 owing to inconsistent data14-17 and a lack of direct supporting evidence. Here we revisited the astrocyte glutamate exocytosis hypothesis by considering the emerging molecular heterogeneity of astrocytes18-21 and using molecular, bioinformatic and imaging approaches, together with cell-specific genetic tools that interfere with glutamate exocytosis in vivo. By analysing existing single-cell RNA-sequencing databases and our patch-seq data, we identified nine molecularly distinct clusters of hippocampal astrocytes, among which we found a notable subpopulation that selectively expressed synaptic-like glutamate-release machinery and localized to discrete hippocampal sites. Using GluSnFR-based glutamate imaging22 in situ and in vivo, we identified a corresponding astrocyte subgroup that responds reliably to astrocyte-selective stimulations with subsecond glutamate release events at spatially precise hotspots, which were suppressed by astrocyte-targeted deletion of vesicular glutamate transporter 1 (VGLUT1). Furthermore, deletion of this transporter or its isoform VGLUT2 revealed specific contributions of glutamatergic astrocytes in cortico-hippocampal and nigrostriatal circuits during normal behaviour and pathological processes. By uncovering this atypical subpopulation of specialized astrocytes in the adult brain, we provide insights into the complex roles of astrocytes in central nervous system (CNS) physiology and diseases, and identify a potential therapeutic target.

Unexpected concentration dependence of the mass accommodation coefficient of water on aqueous triethylene glycol droplets.

The mass accommodation coefficient αM of water on aqueous triethylene glycol droplets was determined for water mole fractions in the range xmol = 0.1-0.93 and temperatures between 21 and 26 °C from modulated Mie scattering measurement on single optically-trapped droplets in combination with a kinetic multilayer model. αM reaches minimum values around 0.005 at a critical water concentration of xmol = 0.38, and increases with decreasing water content to a value of ≈0.1 for almost pure triethylene glycol droplets, essentially independent of the temperature. Above xmol = 0.38, αM first increases with increasing water content and then stabilises at a value of ≈0.1 at the lowest temperatures, while at the highest temperature its value remains around 0.005. We analysed the unexpected concentration and temperature dependence with a previously proposed two-step model for mass accommodation which provides concentration and temperature-dependent activation enthalpies and entropies. We suggest that the unexpected minimum in αM at intermediate water concentrations might arise from a more or less saturated hydrogen-bond network that forms at the droplet surface.

Regulation of the Methylation and Expression Levels of the BMPR2 Gene by SIN3a as a Novel Therapeutic Mechanism in Pulmonary Arterial Hypertension.

BACKGROUND: Epigenetic mechanisms are critical in the pathogenesis of pulmonary arterial hypertension (PAH). Previous studies have suggested that hypermethylation of the BMPR2 (bone morphogenetic protein receptor type 2) promoter is associated with BMPR2 downregulation and progression of PAH. Here, we investigated for the first time the role of SIN3a (switch-independent 3a), a transcriptional regulator, in the epigenetic mechanisms underlying hypermethylation of BMPR2 in the pathogenesis of PAH. METHODS: We used lung samples from PAH patients and non-PAH controls, preclinical mouse and rat PAH models, and human pulmonary arterial smooth muscle cells. Expression of SIN3a was modulated using a lentiviral vector or a siRNA in vitro and a specific adeno-associated virus serotype 1 or a lentivirus encoding for human SIN3a in vivo. RESULTS: SIN3a is a known transcriptional regulator; however, its role in cardiovascular diseases, especially PAH, is unknown. It is interesting that we detected a dysregulation of SIN3 expression in patients and in rodent models, which is strongly associated with decreased BMPR2 expression. SIN3a is known to regulate epigenetic changes. Therefore, we tested its role in the regulation of BMPR2 and found that BMPR2 is regulated by SIN3a. It is interesting that SIN3a overexpression inhibited human pulmonary arterial smooth muscle cells proliferation and upregulated BMPR2 expression by preventing the methylation of the BMPR2 promoter region. RNA-sequencing analysis suggested that SIN3a downregulated the expression of DNA and histone methyltransferases such as DNMT1 (DNA methyltransferase 1) and EZH2 (enhancer of zeste 2 polycomb repressive complex 2) while promoting the expression of the DNA demethylase TET1 (ten-eleven translocation methylcytosine dioxygenase 1). Mechanistically, SIN3a promoted BMPR2 expression by decreasing CTCF (CCCTC-binding factor) binding to the BMPR2 promoter. Last, we identified intratracheal delivery of adeno-associated virus serotype human SIN3a to be a beneficial therapeutic approach in PAH by attenuating pulmonary vascular and right ventricle remodeling, decreasing right ventricle systolic pressure and mean pulmonary arterial pressure, and restoring BMPR2 expression in rodent models of PAH. CONCLUSIONS: All together, our study unveiled the protective and beneficial role of SIN3a in pulmonary hypertension. We also identified a novel and distinct molecular mechanism by which SIN3a regulates BMPR2 in human pulmonary arterial smooth muscle cells. Our study also identified lung-targeted SIN3a gene therapy using adeno-associated virus serotype 1 as a new promising therapeutic strategy for treating patients with PAH.

Charge Effects on the Photodegradation of Single Optically Trapped Oleic Acid Aerosol Droplets.

It has recently been reported that reactions can occur faster in microdroplets than in extended condensed matter. The electric charge of droplets has also been suggested as a possible cause of this phenomenon. Here, we investigate the influence of electric charges on the photodegradation of single, optically trapped oleic acid aerosol droplets in the absence of other reactive species. The temporal evolution of the chemical composition and the size of droplets with charge states ranging from 0 to 104 elementary charges were retrieved from Raman spectra and elastic light scattering, respectively. No influence of the droplet charge was observed, either on the chemical composition or on the kinetics. Based on a kinetic multilayer model, we propose a reaction mechanism with the photoexcitation of oleic acid into an excited state, subsequent decay into intermediates and further photoexcitation of intermediates and their decay into nonvolatile and volatile products.

Senescence of Alveolar Type 2 Cells Drives Progressive Pulmonary Fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) is an insidious and fatal interstitial lung disease associated with declining pulmonary function. Accelerated aging, loss of epithelial progenitor cell function and/or numbers, and cellular senescence are implicated in the pathogenies of IPF.Objectives: We sought to investigate the role of alveolar type 2 (AT2) cellular senescence in initiation and/or progression of pulmonary fibrosis and therapeutic potential of targeting senescence-related pathways and senescent cells.Methods: Epithelial cells of 9 control donor proximal and distal lung tissues and 11 IPF fibrotic lung tissues were profiled by single-cell RNA sequencing to assesses the contribution of epithelial cells to the senescent cell fraction for IPF. A novel mouse model of conditional AT2 cell senescence was generated to study the role of cellular senescence in pulmonary fibrosis.Measurements and Main Results: We show that AT2 cells isolated from IPF lung tissue exhibit characteristic transcriptomic features of cellular senescence. We used conditional loss of Sin3a in adult mouse AT2 cells to initiate a program of p53-dependent cellular senescence, AT2 cell depletion, and spontaneous, progressive pulmonary fibrosis. We establish that senescence rather than loss of AT2 cells promotes progressive fibrosis and show that either genetic or pharmacologic interventions targeting p53 activation or senescence block fibrogenesis.Conclusions: Senescence of AT2 cells is sufficient to drive progressive pulmonary fibrosis. Early attenuation of senescence-related pathways and elimination of senescent cells are promising therapeutic approaches to prevent pulmonary fibrosis.

Sin3a regulates epithelial progenitor cell fate during lung development.

Mechanisms that regulate tissue-specific progenitors for maintenance and differentiation during development are poorly understood. Here, we demonstrate that the co-repressor protein Sin3a is crucial for lung endoderm development. Loss of Sin3a in mouse early foregut endoderm led to a specific and profound defect in lung development with lung buds failing to undergo branching morphogenesis and progressive atrophy of the proximal lung endoderm with complete epithelial loss at later stages of development. Consequently, neonatal pups died at birth due to respiratory insufficiency. Further analysis revealed that loss of Sin3a resulted in embryonic lung epithelial progenitor cells adopting a senescence-like state with permanent cell cycle arrest in G1 phase. This was mediated at least partially through upregulation of the cell cycle inhibitors Cdkn1a and Cdkn2c. At the same time, loss of endodermal Sin3a also disrupted cell differentiation of the mesoderm, suggesting aberrant epithelial-mesenchymal signaling. Together, these findings reveal that Sin3a is an essential regulator for early lung endoderm specification and differentiation.

Selecting for lactic acid producing and utilising bacteria in anaerobic enrichment cultures.

Lactic acid-producing bacteria are important in many fermentations, such as the production of biobased plastics. Insight in the competitive advantage of lactic acid bacteria over other fermentative bacteria in a mixed culture enables ecology-based process design and can aid the development of sustainable and energy-efficient bioprocesses. Here we demonstrate the enrichment of lactic acid bacteria in a controlled sequencing batch bioreactor environment using a glucose-based medium supplemented with peptides and B vitamins. A mineral medium enrichment operated in parallel was dominated by Ethanoligenens species and fermented glucose to acetate, butyrate and hydrogen. The complex medium enrichment was populated by Lactococcus, Lactobacillus and Megasphaera species and showed a product spectrum of acetate, ethanol, propionate, butyrate and valerate. An intermediate peak of lactate was observed, showing the simultaneous production and consumption of lactate, which is of concern for lactic acid production purposes. This study underlines that the competitive advantage for lactic acid-producing bacteria primarily lies in their ability to attain a high biomass specific uptake rate of glucose, which was two times higher for the complex medium enrichment when compared to the mineral medium enrichment. The competitive advantage of lactic acid production in rich media can be explained using a resource allocation theory for microbial growth processes.

Correction: Assessing relative humidity dependent photoacoustics to retrieve mass accommodation coefficients of single optically trapped aerosol particles.

Correction for 'Assessing relative humidity dependent photoacoustics to retrieve mass accommodation coefficients of single optically trapped aerosol particles' by Matus E. Diveky et al., Phys. Chem. Chem. Phys., 2019, 21, 4721-4731, DOI: .

Differences in the Tensor Veli Palatini Muscle and Hearing Status in Children With and Without 22q11.2 Deletion Syndrome.

PURPOSE: To investigate the dimensions of the tensor veli palatini (TVP) muscle using high image resolution 3-dimensional magnetic resonance imaging (MRI) of the soft palate among children with normal velopharyngeal and craniofacial anatomy and to compare values to individuals with a diagnosis of 22q11.2 deletion syndrome (22q11DS). We also sought to determine whether there is a relationship between hypoplasia of the TVP and severity of middle ear dysfunction and hearing loss. METHODS: Three-dimensional MRI were used to collect and analyze data obtained across 53 children between 4 and 12 years of age, including 40 children with normal velopharyngeal and craniofacial anatomy and 13 children with a diagnosis of 22q11.2 DS. Tensor veli palatini muscle length, thickness, and volume as well as bihamular distance were compared among participant groups. RESULTS: A Welch's t-test revealed that the TVP in participants with 22q11DS is significantly shorter (P = .005, 17.3 vs 19.0 mm), thinner (P < .001, 1.1 vs 1.8 mm), and less voluminous (P < .001, 457.5 vs 667.3 mm3) than participants without 22q11DS. Participants with 22q11DS also had a greater (P = .006, 27.7 vs 24.7 mm) bihamular distance than participants without 22q11DS. There was an inverse relationship between TVP abnormalities noted above and the severity of audiologic and otologic histories. CONCLUSION: The TVP muscle is substantially reduced in volume, length, and thickness in children with 22q11DS. These findings serve as preliminary support for the association of patient hearing and otologic severity and TVP dysmorphology.

The chromatin-associated Sin3B protein is required for hematopoietic stem cell functions in mice.

Hematopoietic stem cells (HSCs) reside at the top of the hematopoietic hierarchy and are the origin of all blood cells produced throughout an individual's life. The balance between HSC self-renewal and differentiation is maintained by various intrinsic and extrinsic mechanisms. Among these, the molecular pathways that restrict cell cycle progression are critical to the maintenance of functional HSCs. Alterations in the regulation of cell cycle progression in HSCs invariably lead to the development of hematologic malignancies or bone marrow failure syndromes. Here we report that hematopoietic-specific genetic inactivation of Sin3B, an essential component of the mammalian Sin3-histone deacetylase corepressor complex, severely impairs the competitive repopulation capacity of HSCs. Sin3B-deleted HSCs accumulate and fail to properly differentiate following transplantation. Moreover, Sin3B inactivation impairs HSC quiescence and sensitizes mice to myelosuppressive therapy. Together, these results identify Sin3B as a novel and critical regulator of HSC functions.

Assessing relative humidity dependent photoacoustics to retrieve mass accommodation coefficients of single optically trapped aerosol particles.

Photoacoustic spectroscopy is widely used to measure the light absorption of aerosols. However, the impact of key factors such as the effect of relative humidity and mass exchange on photoacoustic measurements are still poorly understood. We assess such measurement biases and their physical origin by analysing the photoacoustic signal of single tetraethylene glycol (TEG) particles at varying relative humidities. Our results show a decrease in the photoacoustic signal at elevated relative humidities for small particles (0.8-1.5 µm), while for larger sizes (2.2-3.2 µm) the trend is reversed. We model the photoacoustic signal to interpret the observed behaviour in terms of mass and heat flux contribution. The single particle photoacoustic signal analysis presented in this paper additionally allows for the retrieval of the mass accommodation coefficient. Fitting our experimental data to the theoretical model reveals values of αM ≈ 0.02-0.005 for water on TEG in the temperature range 295-309 K.

Standardized ultrasound templates for diagnosing appendicitis reduce annual imaging costs.

BACKGROUND: Ultrasound is preferred over computed tomography (CT) for diagnosing appendicitis in children to avoid undue radiation exposure. We previously reported our experience in instituting a standardized appendicitis ultrasound template, which decreased CT rates by 67.3%. In this analysis, we demonstrate the ongoing cost savings associated with using this template. METHODS: Retrospective chart review for the time period preceding template implementation (June 2012-September 2012) was combined with prospective review through December 2015 for all patients in the emergency department receiving diagnostic imaging for appendicitis. The type of imaging was recorded, and imaging rates and ultrasound test statistics were calculated. Estimated annual imaging costs based on pretemplate ultrasound and CT utilization rates were compared with post-template annual costs to calculate annual and cumulative savings. RESULTS: In the pretemplate period, ultrasound and CT rates were 80.2% and 44.3%, respectively, resulting in a combined annual cost of $300,527.70. Similar calculations were performed for each succeeding year, accounting for changes in patient volume. Using pretemplate rates, our projected 2015 imaging cost was $371,402.86; however, our ultrasound rate had increased to 98.3%, whereas the CT rate declined to 9.6%, yielding an annual estimated cost of $224,853.00 and a savings of $146,549.86. Since implementation, annual savings have steadily increased for a cumulative cost savings of $336,683.83. CONCLUSIONS: Standardizing ultrasound reports for appendicitis not only reduces the use of CT scans and the associated radiation exposure but also decreases annual imaging costs despite increased numbers of imaging studies. Continued cost reduction may be possible by using diagnostic algorithms.

Technical Considerations for Nephron-Sparing Surgery in Children: What Is Needed to Preserve Renal Units?

BACKGROUND: Chemotherapy is used preoperatively for children with bilateral Wilms tumor (BWT) or unilateral high-risk Wilms tumor (UHRWT) to promote tumor regression to facilitate renal preservation with nephron-sparing surgery (NSS). In adults, various surgical techniques have been described to preserve renal tissue. Few studies have examined the use of surgical adjuncts in NSS in children with renal tumors. METHODS: We performed a multi-institutional retrospective review of patients with BWT or UHRWT. Patient demographics, tumor size at diagnosis, following neoadjuvant chemotherapy, utilization of surgical adjuncts including intraoperative ultrasound (IOUS), margin status, complications, renal function, and follow-up were recorded. RESULTS: The cohort comprised 23 patients: 18 BWT, 3 UHRWT, and 2 patients with solitary kidney. Twenty-two of the 23 patients had successful NSS. IOUS was used 19 times, and seven had positive margins after surgery. Cooling/vascular isolation was used six times. At a median follow-up of 18 mo, median estimated glomerular filtration rate Schwartz was 126 mL/min/1.73 m2 and median serum creatinine 0.39 mg/dL in the 22 patients who had successful NSS. There have been no tumor recurrences. CONCLUSIONS: In patients with BWT and UHRWT, surgical adjuncts such as cooling/vascular isolation are uncommonly performed. IOUS may be helpful but does not guarantee negative microscopic margins. LEVEL OF EVIDENCE: Level 4, Case series with no comparison group.

Phase transition dynamics of single optically trapped aqueous potassium carbonate particles.

Fast dynamics (down to 10 ms) during exposure to changing relative humidity of single optically trapped K2CO3 particles were observed in the submicron to micron size range with time-resolved broadband light scattering and Raman spectroscopy. The study shows that complex multiple processes accompany efflorescence and deliquescence of unsupported aerosol particles. Efflorescence can occur in a single process in less than 10 ms (prompt) or proceed via multiple successive processes (multistep) that can last up to more than 10 seconds. The efflorescence relative humidity lies in the range of 9 to 25%. Raman spectra of the effloresced particles reveal that the final state of the particle is independent of the pathway. Deliquescence cycles start with an initial uptake of water followed by multiple complex processes which end at the deliquescence relative humidity (44-50%). The study reveals that complex multiple processes during phase transitions are not limited to deposited particles where heterogeneous processes may occur or large particles in the upper micrometer size range as previously observed.

Structural insights into the assembly of the histone deacetylase-associated Sin3L/Rpd3L corepressor complex.

Acetylation is correlated with chromatin decondensation and transcriptional activation, but its regulation by histone deacetylase (HDAC)-bearing corepressor complexes is poorly understood. Here, we describe the mechanism of assembly of the mammalian Sin3L/Rpd3L complex facilitated by Sds3, a conserved subunit deemed critical for proper assembly. Sds3 engages a globular, helical region of the HDAC interaction domain (HID) of the scaffolding protein Sin3A through a bipartite motif comprising a helix and an adjacent extended segment. Sds3 dimerizes through not only one of the predicted coiled-coil motifs but also, the segment preceding it, forming an ∼ 150-Å-long antiparallel dimer. Contrary to previous findings in yeast, Sin3A rather than Sds3 functions in recruiting HDAC1 into the complex by engaging the latter through a highly conserved segment adjacent to the helical HID subdomain. In the resulting model for the ternary complex, the two copies of the HDACs are situated distally and dynamically because of a natively unstructured linker connecting the dimerization domain and the Sin3A interaction domain of Sds3; these features contrast with the static organization described previously for the NuRD (nucleosome remodeling and deacetylase) complex. The Sds3 linker features several conserved basic residues that could potentially maintain the complex on chromatin by nonspecific interactions with DNA after initial recruitment by sequence-specific DNA-binding repressors.

URI Regulates KAP1 Phosphorylation and Transcriptional Repression via PP2A Phosphatase in Prostate Cancer Cells.

URI (unconventional prefoldin RPB5 interactor protein) is an unconventional prefoldin, RNA polymerase II interactor that functions as a transcriptional repressor and is part of a larger nuclear protein complex. The components of this complex and the mechanism of transcriptional repression have not been characterized. Here we show that KAP1 (KRAB-associated protein 1) and the protein phosphatase PP2A interact with URI. Mechanistically, we show that KAP1 phosphorylation is decreased following recruitment of PP2A by URI. We functionally characterize the novel URI-KAP1-PP2A complex, demonstrating a role of URI in retrotransposon repression, a key function previously demonstrated for the KAP1-SETDB1 complex. Microarray analysis of annotated transposons revealed a selective increase in the transcription of LINE-1 and L1PA2 retroelements upon knockdown of URI. These data unveil a new nuclear function of URI and identify a novel post-transcriptional regulation of KAP1 protein that may have important implications in reactivation of transposable elements in prostate cancer cells.

Ultraviolet broadband light scattering for optically-trapped submicron-sized aerosol particles.

We describe a broadband light scattering setup for the characterization of size and refractive index of single submicron-to-micron sized aerosol particles. Individual particles are isolated in air by a quadruple Bessel beam optical trap or a counter-propagating optical tweezer. The use of very broadband radiation in the wavelength range from 320 to 700 nm covering the ultraviolet region allows to size submicron particles. We show that a broad wavelength range is required to determine the particle radius and the refractive index with an uncertainty of several nanometers and ∼ 0.01, respectively. The smallest particle radius that can be accurately determined lies around 300 nm. Wavelength-dependent refractive index data over a broad range are obtained, including the ultraviolet region where corresponding data are rare. Four different applications are discussed: (1) the sizing of submicron polystyrene latex spheres, (2) the evaporation of binary glycerol water droplets, (3) hydration/dehydration cycling of aqueous potassium carbonate droplets, and (4) photochemical reactions of oleic acid droplets.

Inhibition of androgen receptor and ß-catenin activity in prostate cancer.

Androgen receptor (AR) is the major therapeutic target in aggressive prostate cancer. However, targeting AR alone can result in drug resistance and disease recurrence. Therefore, simultaneous targeting of multiple pathways could in principle be an effective approach to treating prostate cancer. Here we provide proof-of-concept that a small-molecule inhibitor of nuclear ß-catenin activity (called C3) can inhibit both the AR and ß-catenin-signaling pathways that are often misregulated in prostate cancer. Treatment with C3 ablated prostate cancer cell growth by disruption of both ß-catenin/T-cell factor and ß-catenin/AR protein interaction, reflecting the fact that T-cell factor and AR have overlapping binding sites on ß-catenin. Given that AR interacts with, and is transcriptionally regulated by ß-catenin, C3 treatment also resulted in decreased occupancy of ß-catenin on the AR promoter and diminished AR and AR/ß-catenin target gene expression. Interestingly, C3 treatment resulted in decreased AR binding to target genes accompanied by decreased recruitment of an AR and ß-catenin cofactor, coactivator-associated arginine methyltransferase 1 (CARM1), providing insight into the unrecognized function of ß-catenin in prostate cancer. Importantly, C3 inhibited tumor growth in an in vivo xenograft model and blocked renewal of bicalutamide-resistant sphere-forming cells, indicating the therapeutic potential of this approach.

Stability of aerosol droplets in Bessel beam optical traps under constant and pulsed external forces.

We report on the dynamics of aerosol droplets in optical traps under the influence of additional constant and pulsed external forces. Experimental results are compared with simulations of the three-dimensional droplet dynamics for two types of optical traps, the counter-propagating Bessel beam (CPBB) trap and the quadruple Bessel beam (QBB) trap. Under the influence of a constant gas flow (constant external force), the QBB trap is found to be more stable compared with the CPBB trap. By contrast, under pulsed laser excitation with laser pulse durations of nanoseconds (pulsed external force), the type of trap is of minor importance for the droplet stability. It typically needs pulsed laser forces that are several orders of magnitude higher than the optical forces to induce escape of the droplet from the trap. If the droplet strongly absorbs the pulsed laser light, these escape forces can be strongly reduced. The lower stability of absorbing droplets is a result of secondary thermal processes that cause droplet escape.