There are currently no functional neuromuscular junction (hNMJ) systems composed of human cells that could be used for drug evaluations or toxicity testing in vitro. These systems are needed to ...evaluate NMJs for diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy or other neurodegenerative diseases or injury states. There are certainly no model systems, animal or human, that allows for isolated treatment of motoneurons or muscle capable of generating dose response curves to evaluate pharmacological activity of these highly specialized functional units. A system was developed in which human myotubes and motoneurons derived from stem cells were cultured in a serum-free medium in a BioMEMS construct. The system is composed of two chambers linked by microtunnels to enable axonal outgrowth to the muscle chamber that allows separate stimulation of each component and physiological NMJ function and MN stimulated tetanus. The muscle's contractions, induced by motoneuron activation or direct electrical stimulation, were monitored by image subtraction video recording for both frequency and amplitude. Bungarotoxin, BOTOX® and curare dose response curves were generated to demonstrate pharmacological relevance of the phenotypic screening device. This quantifiable functional hNMJ system establishes a platform for generating patient-specific NMJ models by including patient-derived iPSCs.
•A system in which stem cell derived human myotubes and MNs were cultured in a serum-free medium in a BioMEMS construct.•Directed axonal outgrowth links muscle and MN chambers to allow for physiological NMJ function and MN stimulated tetanus.•The muscle’s contractions were monitored by image subtraction video recording for both frequency and amplitude.•Bungarotoxin, BOTOX® and curare dose response curves were generated for pharmacological relevance of the phenotypic screen.•This hNMJ system establishes a platform for generating patient-specific models using iPSCs for personalized medicine.
Abstract
Alternative pre-mRNA splicing decisions are regulated by RNA binding proteins (RBPs) that can activate or repress regulated splice sites. Repressive RBPs typically harness multivalent ...interactions to bind stably to target RNAs. Multivalency can be achieved by homomeric oligomerization and heteromeric interactions with other RBPs, often mediated by intrinsically disordered regions (IDRs), and by possessing multiple RNA binding domains. Cell-specific splicing decisions often involve the action of widely expressed RBPs, which are able to bind multivalently around target exons, but without effect in the absence of a cell-specific regulator. To address how cell-specific regulators can collaborate with constitutive RBPs in alternative splicing regulation, we used the smooth-muscle specific regulator RBPMS. Recombinant RBPMS is sufficient to confer smooth muscle cell specific alternative splicing of Tpm1 exon 3 in cell-free assays by preventing assembly of ATP-dependent splicing complexes. This activity depends upon a C-terminal IDR that facilitates dynamic higher-order self-assembly, cooperative binding to multivalent RNA and interactions with widely expressed splicing co-regulators, including MBNL1 and RBFOX2, allowing cooperative assembly of stable cell-specific regulatory complexes.
Graphical Abstract
Graphical Abstract
Histone chaperoning ensures genomic integrity during routine processes such as DNA replication and transcription as well as DNA repair upon damage. Here, we identify a nuclear J domain protein, Dnj4, ...in the fungal pathogen Cryptococcus neoformans and demonstrate that it interacts with histones 3 and 4, suggesting a role as a histone chaperone. In support of this idea, a
deletion mutant had elevated levels of DNA damage and was hypersensitive to DNA-damaging agents. The transcriptional response to DNA damage was also impaired in the
mutant. Genes related to DNA damage and iron homeostasis were upregulated in the wild-type strain in response to hydroxyurea treatment; however, their upregulation was either absent from or reduced in the
mutant. Accordingly, excess iron rescued the mutant's growth in response to DNA-damaging agents. Iron homeostasis is crucial for virulence in C. neoformans; however, Dnj4 was found to be dispensable for disease in a mouse model of cryptococcosis. Finally, we confirmed a conserved role for Dnj4 as a histone chaperone by expressing it in Saccharomyces cerevisiae and showing that it disrupted endogenous histone chaperoning. Altogether, this study highlights the importance of a JDP cochaperone in maintaining genome integrity in C. neoformans.
DNA replication, gene expression, and genomic repair all require precise coordination of the many proteins that interact with DNA. This includes the histones as well as their chaperones. In this study, we show that a histone chaperone, Dnj4, is required for genome integrity and for the response to DNA damage. The gene encoding this protein in Cryptococcus neoformans lacks an ortholog in Saccharomyces cerevisiae; however, it is conserved in humans in which its ortholog is essential. Since it is not essential in C. neoformans, we were able to generate deletion mutants to characterize the roles of Dnj4. We also expressed Dnj4 in S. cerevisiae, in which it was able to bind S. cerevisiae histones and interfere with existing histone chaperoning machinery. Therefore, we show a conserved role for Dnj4 in histone chaperoning that suggests that C. neoformans is useful to better understand aspects of this important biological process.
Abstract Voigt profile (VP) decomposition of quasar absorption lines is key to studying intergalactic gas and the baryon cycle governing the formation and evolution of galaxies. The VP velocities, ...column densities, and Doppler b parameters inform us of the kinematic, chemical, and ionization conditions of these astrophysical environments. A drawback of traditional VP fitting is that it can be human-time intensive. With the coming next generation of large all-sky survey telescopes with multiobject high-resolution spectrographs, the time demands will significantly outstrip our resources. Deep learning pipelines hold the promise to keep pace and deliver science-digestible data products. We explore the application of deep learning convolutional neural networks (CNNs) for predicting VP-fitted parameters directly from the normalized pixel flux values in quasar absorption line profiles. A CNN was applied to 56 single-component Mg ii λ λ 2796, 2803 doublet absorption line systems observed with HIRES and UVES ( R = 45,000). The CNN predictions were statistically indistinct from those of a traditional VP fitter. The advantage is that, once trained, the CNN processes systems ∼10 5 times faster than a human expert fitting VP profiles by hand. Our pilot study shows that CNNs hold promise to perform bulk analysis of quasar absorption line systems in the future.
Evolutionary theory predicts that reproduction entails costs that detract from somatic maintenance, accelerating biological aging. Despite support from studies in human and non-human animals, ...mechanisms linking 'costs of reproduction' (CoR) to aging are poorly understood. Human pregnancy is characterized by major alterations in metabolic regulation, oxidative stress, and immune cell proliferation. We hypothesized that these adaptations could accelerate blood-derived cellular aging. To test this hypothesis, we examined gravidity in relation to telomere length (TL, n = 821) and DNA-methylation age (DNAmAge, n = 397) in a cohort of young (20-22 year-old) Filipino women. Age-corrected TL and accelerated DNAmAge both predict age-related morbidity and mortality, and provide markers of mitotic and non-mitotic cellular aging, respectively. Consistent with theoretical predictions, TL decreased (p = 0.031) and DNAmAge increased (p = 0.007) with gravidity, a relationship that was not contingent upon resource availability. Neither biomarker was associated with subsequent fertility (both p > 0.3), broadly consistent with a causal effect of gravidity on cellular aging. Our findings provide evidence that reproduction in women carries costs in the form of accelerated aging through two independent cellular pathways.
Aminosilica materials are promising candidates for CO2 capture from dilute streams such as ambient air and flue gas. Most aminosilica sorbents are constructed using primary and/or secondary amines, ...which have been shown to primarily react with CO2 to form alkylammonium carbamates and related structures. While ammonium bicarbonate formation is known to occur in aqueous amine solutions in the presence of CO2, there has been conflicting evidence of its formation on solid supported analogues. To probe if the ammonium bicarbonate species can exist on solid supported amines, tertiary amines, which are known to form bicarbonates in aqueous solution, are grafted onto mesoporous silica SBA-15, and the materials are further characterized using in situ FTIR spectroscopy and solid-state NMR spectroscopy in the presence of humid and dry CO2. Dry and humid CO2 capacities for these sorbents are also evaluated using fixed bed experiments and thermogravimetric analysis. This work shows that ammonium bicarbonates can exist on solid supported amines but also demonstrates that tertiary amines are poor CO2 sorbents under the conditions employed.
Background Paro and other robot animals can improve wellbeing for older adults and people with dementia, through reducing depression, agitation and medication use. However, nursing and care staff we ...contacted expressed infection control concerns. Little related research has been published. We assessed (i) how microbiologically contaminated robot animals become during use by older people within a care home and (ii) efficacy of a cleaning procedure. Methods This study had two stages. In stage one we assessed microbial load on eight robot animals after interaction with four care home residents, and again following cleaning by a researcher. Robot animals provided a range of shell-types, including fur, soft plastic, and solid plastic. Stage two involved a similar process with two robot animals, but a care staff member conducted cleaning. The cleaning process involved spraying with anti-bacterial product, brushing fur-type shells, followed by vigorous top-to-tail cleaning with anti-bacterial wipes on all shell types. Two samples were taken from each of eight robots in stage one and two robots in stage two (20 samples total). Samples were collected using contact plate stamping and evaluated using aerobic colony count and identification (gram stain, colony morphology, coagulase agglutination). Colony counts were measured by colony forming units per square centimetre (CFU/cm.sup.2). Results Most robots acquired microbial loads well above an acceptable threshold of 2.5 CFU/cm.sup.2 following use. The bacteria identified were micrococcus species, coagulase negative staphylococcus, diptheriods, aerobic spore bearers, and staphylococcus aureus, all of which carry risk for human health. For all devices the CFU/cm.sup.2 reduced to well within accepted limits following cleaning by both researcher and care staff member. Conclusions Companion robots will acquire significant levels of bacteria during normal use. The simple cleaning procedure detailed in this study reduced microbial load to acceptable levels in controlled experiments. Further work is needed in the field and to check the impact on the transmission of viruses.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
While recent years have seen many analyses of techniques to reduce urban heat island, nearly all of these studies have either been evaluations of real small scale applications or attempts to model ...the effects of large scale applications. This study is an attempt to analyze a real large scale application by observing recent vegetated and reflective surfaces in LANDSAT images of Chicago, a city which has deployed a variety of heat island combative methods over the last 15 years. Results show that Chicago's new reflective surfaces since 1995 produced a noticeable impact on the citywide albedo, raising it by about 0.016, while citywide NDVI increase is around 0.007. This finding along with counts of pixels with increased albedo and NDVI suggest that the reflective strategies influenced a larger area of the city than the vegetative methods. Additionally, plots between albedo increase and corresponding LANDSAT temperature change over the test period have linear regressions with steeper slopes (-15.7) and stronger linear correlations (-0.33) than plots between NDVI increase and temperature change (-8.9 slope, -0.17 correlation). This indicates that the albedo increases produced greater LANDSAT cooling than the NDVI increases. Observation of aerial images confirmed that typical instances of efforts to increase albedo, such as reflective roofs, produced stronger LANDSAT cooling than common instances of NDVI efforts, such as green roofs, street trees and green spaces. Accordingly, the reflective strategies were likely much more effective at cooling Chicago's LANDSAT heat island and may signify a generally more effective strategy for similar cities.
Objective
To compare the detection rates of prostate cancer between systematic biopsy and targeted biopsy using a stereotactic robot‐assisted transperineal prostate platform.
Materials and Methods
We ...identified consecutive patients with suspicious lesion(s) on multiparametric magnetic resonance imaging (mpMRI), who underwent both systematic and MRI‐transrectal ultrasonography (US) fusion targeted biopsy using our proprietary transperineal robot‐assisted prostate biopsy platform between January 2015 and January 2019 at our institution, for retrospective analysis. Comparative analysis was performed between systematic and targeted biopsy using McNemar’s test, and the cohort was further stratified by prior biopsy status and Prostate Imaging Reporting and Data System (PI‐RADS) v2.0 score. International Society of Urological Pathology (ISUP) grade group (GG) ≥2 cancers (previously known as Gleason grade ≥7) were considered to be clinically significant.
Results
A total of 500 patients were included in our final analysis, of whom 67 (13%) were patients with low‐risk cancer on active surveillance. Of the 433 patients without prior diagnosis of cancer, 288 (67%) were biopsy‐naïve. A total of 248 (57%) were diagnosed with prostate cancer, with 199 (46%) having clinically significant prostate cancer (ISUP GG ≥2). There were no statistically significant differences in the overall prostate cancer and clinically significant prostate cancer detection rate between systematic and targeted biopsy (51% vs 49% and 40% vs 38% respectively; P = 0.306 and P = 0.609). Of the 248 prostate cancers detected, 75% (187/248) were detected on both systematic and targeted biopsy, 14% (35/248) were detected on systematic biopsy alone and 11% (26/248) were detected on targeted biopsy alone. Of the 199 clinically significant cancers detected, 69% (138/199) were detected on both systematic and targeted biopsy, 17% (33/199) on systematic biopsy alone and 14% (28/199) on targeted biopsy alone. There were no statistically significant differences in the detection rate between systematic and targeted biopsy for both overall and clinically significant prostate cancer, even when the cohort was stratified by prior biopsy status and PI‐RADS score. Targeted biopsy has greater sampling efficiency compared to systematic biopsy for both overall and clinically significant prostate cancer (23.2% vs 9.8%, P < 0.001 and 14.8% vs 5.6%, P < 0.001).
Conclusions
Using our robot‐assisted transperineal prostate platform, combined MRI‐US targeted biopsy with concurrent systematic prostate systematic biopsy probably represents the optimal method for the detection of clinically significant prostate cancer.
A dibromo derivative of Dewar benzene, trans-5,6-dibromobicyclo2.2.0hex-1-ene, was polymerized using ring-opening metathesis polymerization (ROMP). The reaction proceeded in a controlled manner as ...changing the initial monomer-to-catalyst ratio afforded monodispersed polymers with tunable molecular weights and growing polymer chains were extended upon subsequent exposure to additional monomer. Treatment of the halogenated polymers with an alkyllithium reagent resulted in elimination followed by isomerization to afford trans-poly(acetylene). Based on a series of mechanistic and spectroscopic studies, the transformation was proposed to proceed through a cyclobutenyl intermediate that undergoes rearrangement. The methodology was found to be versatile as triblock copolymers containing the halogenated homopolymer were prepared and converted to their poly(acetylene)-containing derivatives. The polymers were characterized using gel permeation chromatography as well as a range of spectroscopic (NMR, FT-IR, UV–vis, and Raman) and analytical techniques.
PDF
