A Hapa Family in Chekhov\u27s Three Sisters

It is an act of courage or foolhardiness to produce theatre in the heart of the film world, depending on your point of view and how large the houses turn out to be. In the fall of 2005, I produced Three Sisters in a 60-seat theatre in Burbank, California (home of Disney and Warner Bros.). The odds were stacked even higher against the show\u27s success when I stipulated that the main characters, the upper-class and highly educated Russian Prozorov siblings, had to be played by hapa actors. This essay describes my attempt to interpret the play through a multi-ethnic lens while working with a monoracial director, and the challenges this posed, both on the stage and off

Quantifying nuclear wide chromatin compaction by phasor analysis of histone Förster resonance energy transfer (FRET) in frequency domain fluorescence lifetime imaging microscopy (FLIM) data.

The nanometer spacing between nucleosomes throughout global chromatin organisation modulates local DNA template access, and through continuous dynamic rearrangements, regulates genome function [1]. However, given that nucleosome packaging occurs on a spatial scale well below the diffraction limit, real time observation of chromatin structure in live cells by optical microscopy has proved technically difficult, despite recent advances in live cell super resolution imaging [2]. One alternative solution to quantify chromatin structure in a living cell at the level of nucleosome proximity is to measure and spatially map Förster resonance energy transfer (FRET) between fluorescently labelled histones - the core protein of a nucleosome [3]. In recent work we established that the phasor approach to fluorescence lifetime imaging microscopy (FLIM) is a robust method for the detection of histone FRET which can quantify nuclear wide chromatin compaction in the presence of cellular autofluorescence [4]. Here we share FLIM data recording histone FRET in live cells co-expressing H2B-eGFP and H2B-mCherry. The data was acquired in the frequency domain [5] and processed by the phasor approach to lifetime analysis [6]. The data can be valuable to researchers interested in using the histone FRET assay since it highlights the impact of cellular autofluorescence and acceptor-donor ratio on quantifying chromatin compaction. The data is related to the research article "Phasor histone FLIM-FRET microscopy quantifies spatiotemporal rearrangement of chromatin architecture during the DNA damage response" [4]

Urbanisation and health in China.

China has seen the largest human migration in history, and the country's rapid urbanisation has important consequences for public health. A provincial analysis of its urbanisation trends shows shifting and accelerating rural-to-urban migration across the country and accompanying rapid increases in city size and population. The growing disease burden in urban areas attributable to nutrition and lifestyle choices is a major public health challenge, as are troubling disparities in health-care access, vaccination coverage, and accidents and injuries in China's rural-to-urban migrant population. Urban environmental quality, including air and water pollution, contributes to disease both in urban and in rural areas, and traffic-related accidents pose a major public health threat as the country becomes increasingly motorised. To address the health challenges and maximise the benefits that accompany this rapid urbanisation, innovative health policies focused on the needs of migrants and research that could close knowledge gaps on urban population exposures are needed

In vivo measurement of afferent activity with axon-specific calcium imaging.

In vivo calcium imaging from axons provides direct interrogation of afferent neural activity, informing the neural representations that a local circuit receives. Unlike in somata and dendrites, axonal recording of neural activity-both electrically and optically-has been difficult to achieve, thus preventing comprehensive understanding of neuronal circuit function. Here we developed an active transportation strategy to enrich GCaMP6, a genetically encoded calcium indicator, uniformly in axons with sufficient brightness, signal-to-noise ratio, and photostability to allow robust, structure-specific imaging of presynaptic activity in awake mice. Axon-targeted GCaMP6 enables frame-to-frame correlation for motion correction in axons and permits subcellular-resolution recording of axonal activity in previously inaccessible deep-brain areas. We used axon-targeted GCaMP6 to record layer-specific local afferents without contamination from somata or from intermingled dendrites in the cortex. We expect that axon-targeted GCaMP6 will facilitate new applications in investigating afferent signals relayed by genetically defined neuronal populations within and across specific brain regions

Neutral, water-soluble poly(ester amide) hydrogels for cell encapsulation

© 2020 Elsevier Ltd Hydrogels are of significant interest for cell encapsulation and delivery in regenerative medicine. Poly(ester amide)s (PEAs) are a class of biodegradable polymers that exhibit promise for biomedical applications due to the degradability of the ester and amide linkages in their backbones, their preparation from biomolecules such as amino acids, and the ability to readily tune their properties through a modular synthesis approach. Water-soluble PEAs containing cationic arginine moieties have previously been developed, but to the best of our knowledge, neutral water-soluble PEAs based on non-charged amino acids have not been reported. Using a poly(ethylene glycol) (PEG)-based macromonomer, we describe here the syntheses of water-soluble amino acid-containing PEAs containing crosslinkable alkenes in their backbones. These PEAs were converted into hydrogels through photoinitiated crosslinking and their properties were compared, including gel content, water content, swelling, and Young\u27s moduli. Subsequent cell culture studies on a subset of hydrogels confirmed that human adipose-derived stromal cells (ASCs) showed \u3e 75% viability at 24 h post-encapsulation. To explore the potential of the hydrogels as cell delivery systems for applications in soft tissue regeneration, adipogenic differentiation of the encapsulated ASCs was probed in vitro at 7 days. Analysis of glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity and intracellular lipid accumulation indicated that the hydrogels provided a supportive environment for ASC adipogenesis. Overall, these PEAs provide a new platform that warrants further development for regenerative medicine applications

Cartilage-specific ablation of site-1 protease in mice results in the endoplasmic reticulum entrapment of type IIB procollagen and down-regulation of cholesterol and lipid homeostasis

The proprotein convertase site-1 protease (S1P) converts latent ER-membrane bound transcription factors SREBPs and ATF6 to their active forms. SREBPs are involved in cholesterol and fatty acid homeostasis whereas ATF6 is involved in unfolded protein response pathways (UPR). Cartilage-specific ablation of S1P in mice (S1Pcko) results in abnormal cartilage devoid of type II collagen protein (Col II). S1Pcko mice also lack endochondral bone development. To analyze S1Pcko cartilage we performed double-labeled immunofluorescence studies for matrix proteins that demonstrated that type IIB procollagen is trapped inside the ER in S1Pcko chondrocytes. This retention is specific to type IIB procollagen; other cartilage proteins such as type IIA procollagen, cartilage oligomeric matrix protein (COMP) and aggrecan are not affected. The S1Pcko cartilage thus exhibits COMP-, aggrecan-, and type IIA procollagen-derived matrices but is characterized by the absence of a type IIB procollagen-derived matrix. To understand the molecular reason behind S1Pcko phenotypes we performed genome-wide transcriptional profiling of cartilage isolated from S1Pcko and wild type littermates. While the UPR pathways are unaffected, the SREBPs-directed cholesterol and fatty acid pathways are significantly down-regulated in S1Pcko chondrocytes, with maximal down-regulation of the stearoyl-CoA desaturase-1 (Scd1) gene. However, mouse models that lack Scd1 or exhibit reduction in lipid homeostasis do not suffer from the ER retention of Col II or lack endochondral bone. These studies indicate an indispensable role for S1P in type IIB procollagen trafficking from the ER. This role appears not to be related to lipid pathways or other current known functions of S1P and is likely dependent on additional, yet unknown, S1P substrates in chondrocytes

Canine olfactory ensheathing cells from the olfactory mucosa can be engineered to produce active chondroitinase ABC

A multitude of factors must be overcome following spinal cord injury (SCI) in order to achieve clinical improvement in patients. It is thought that by combining promising therapies these diverse factors could be combatted with the aim of producing an overall improvement in function. Chondroitin sulphate proteoglycans (CSPGs) present in the glial scar that forms following SCI present a significant block to axon regeneration. Digestion of CSPGs by chondroitinase ABC (ChABC) leads to axon regeneration, neuronal plasticity and functional improvement in preclinical models of SCI. However, the enzyme activity decays at body temperature within 24–72 h, limiting the translational potential of ChABC as a therapy. Olfactory ensheathing cells (OECs) have shown huge promise as a cell transplant therapy in SCI. Their beneficial effects have been demonstrated in multiple small animal SCI models as well as in naturally occurring SCI in canine patients. In the present study, we have genetically modified canine OECs from the mucosa to constitutively produce enzymatically active ChABC. We have developed a lentiviral vector that can deliver a mammalian modified version of the ChABC gene to mammalian cells, including OECs. Enzyme production was quantified using the Morgan-Elson assay that detects the breakdown products of CSPG digestion in cell supernatants. We confirmed our findings by immunolabelling cell supernatant samples using Western blotting. OECs normal cell function was unaffected by genetic modification as demonstrated by normal microscopic morphology and the presence of the low affinity neurotrophin receptor (p75NGF) following viral transduction. We have developed the means to allow production of active ChABC in combination with a promising cell transplant therapy for SCI repair

Transesterification of Poly(ethyl glyoxylate): A Route to Structurally Diverse Polyglyoxylates

Polyglyoxylates are a class of self-immolative polymers that depolymerize in solution and the solid state. The glyoxylic acid degradation product is a metabolite in the glyoxylate cycle and can also be processed in the liver in humans, making polyglyoxylates attractive for applications in the environment and in medicine. Although expanding the scope of available polyglyoxylates would enable new properties and applications, highly pure glyoxylate monomers are required for polymerization, and this level of purity is difficult to achieve for many potential monomers. To address this challenge, we report here the 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD)-catalyzed post-polymerization transesterification of poly(ethyl glyoxylate) (PEtG) as a general method for the synthesis of directly inaccessible polyglyoxylates. Using a new end-capping strategy, PEtG compatible with the transesterification reaction was developed. n-Propanol, i-propanol, n-butanol, t-butanol, n-pentanol, n-hexanol, n-octanol, and benzyl alcohol were employed and the reactivities of these different alcohols were investigated. The resulting polyglyoxylates were characterized chemically and their thermal properties were compared. In all cases, the transesterified polyglyoxylates retained the stimuli-responsive depolymerization properties of the parent PEtG. In addition, functional polyglyoxylates based on allyl, propargyl, and furfuryl esters, which are suitable for subsequent click reactions, were prepared. The propargyl-functionalized polyglyoxylate was used to conjugate pyrene, and the resulting molecules underwent a change in fluorescence properties upon depolymerization