Single-cell multiplex chromatin and RNA interactions in ageing human brain.

Dynamically organized chromatin complexes often involve multiplex chromatin interactions and sometimes chromatin-associated RNA1-3. Chromatin complex compositions change during cellular differentiation and ageing, and are expected to be highly heterogeneous among terminally differentiated single cells4-7. Here we introduce the multinucleic acid interaction mapping in single cells (MUSIC) technique for concurrent profiling of multiplex chromatin interactions, gene expression and RNA-chromatin associations within individual nuclei. When applied to 14 human frontal cortex samples from older donors, MUSIC delineated diverse cortical cell types and states. We observed that nuclei exhibiting fewer short-range chromatin interactions were correlated with both an 'older' transcriptomic signature and Alzheimer's disease pathology. Furthermore, the cell type exhibiting chromatin contacts between cis expression quantitative trait loci and a promoter tends to be that in which these cis expression quantitative trait loci specifically affect the expression of their target gene. In addition, female cortical cells exhibit highly heterogeneous interactions between XIST non-coding RNA and chromosome X, along with diverse spatial organizations of the X chromosomes. MUSIC presents a potent tool for exploration of chromatin architecture and transcription at cellular resolution in complex tissues.

Revealing protein-protein interactions at the transcriptome scale by sequencing.

We describe PROPER-seq (protein-protein interaction sequencing) to map protein-protein interactions (PPIs) en masse. PROPER-seq first converts transcriptomes of input cells into RNA-barcoded protein libraries, in which all interacting protein pairs are captured through nucleotide barcode ligation, recorded as chimeric DNA sequences, and decoded at once by sequencing and mapping. We applied PROPER-seq to human embryonic kidney cells, T lymphocytes, and endothelial cells and identified 210,518 human PPIs (collected in the PROPER v.1.0 database). Among these, 1,365 and 2,480 PPIs are supported by published co-immunoprecipitation (coIP) and affinity purification-mass spectrometry (AP-MS) data, 17,638 PPIs are predicted by the prePPI algorithm without previous experimental validation, and 100 PPIs overlap human synthetic lethal gene pairs. In addition, four previously uncharacterized interaction partners with poly(ADP-ribose) polymerase 1 (PARP1) (a critical protein in DNA repair) known as XPO1, MATR3, IPO5, and LEO1 are validated in vivo. PROPER-seq presents a time-effective technology to map PPIs at the transcriptome scale, and PROPER v.1.0 provides a rich resource for studying PPIs.

Rapid 3D Bioprinting of Glioblastoma Model Mimicking Native Biophysical Heterogeneity.

Glioblastoma multiforme (GBM) is the most lethal primary brain tumor characterized by high cellular and molecular heterogeneity, hypervascularization, and innate drug resistance. Cellular components and extracellular matrix (ECM) are the two primary sources of heterogeneity in GBM. Here, biomimetic tri-regional GBM models with tumor regions, acellular ECM regions, and an endothelial region with regional stiffnesses patterned corresponding to the GBM stroma, pathological or normal brain parenchyma, and brain capillaries, are developed. Patient-derived GBM cells, human endothelial cells, and hyaluronic acid derivatives are used to generate a species-matched and biochemically relevant microenvironment. This in vitro study demonstrates that biophysical cues are involved in various tumor cell behaviors and angiogenic potentials and promote different molecular subtypes of GBM. The stiff models are enriched in the mesenchymal subtype, exhibit diffuse invasion of tumor cells, and induce protruding angiogenesis and higher drug resistance to temozolomide. Meanwhile, the soft models demonstrate enrichment in the classical subtype and support expansive cell growth. The three-dimensional bioprinting technology utilized in this study enables rapid, flexible, and reproducible patient-specific GBM modeling with biophysical heterogeneity that can be employed by future studies as a tunable system to interrogate GBM disease mechanisms and screen drug compounds.

Does the presence of more features in a bound representation in working memory require extra object-based attention?

Recent studies have examined the role of attention in retaining bound representations in working memory (WM) and found that object-based attention plays a pivotal role. However, no study has investigated whether maintaining bound representations with more features in WM requires extra object-based attention. We investigated this by examining whether a secondary task consuming object-based attention was more disruptive to the maintenance of bindings in WM when more features were stored per object. We instructed participants to memorize three bound representations in a WM task while manipulating the number of features (two vs. three features) contained in each representation. Moreover, we manipulated whether a secondary task consuming object-based attention was interpolated into the maintenance phase of WM. If extra object-based attention was required after the addition of an extra feature in the bound representation, the secondary task would result in a greater disruption of the three- rather than two-featured binding. In two experiments, we found that the added secondary task significantly impaired the binding performance, but the performance of the two- and three-featured bindings was disrupted to the same extent. These results suggest that the presence of more features in a bound representation in WM does not require extra object-based attention.

Object-based attention in retaining binding in working memory: Influence of activation states of working memory.

It has been suggested that retaining bindings in working memory (WM) requires more object-based attention than retaining constituent features. Recent studies have found that when memorized stimuli are presented sequentially, the most recent stimulus is in a highly accessible privileged state such that it is retained in a relatively automatic and resource-free manner, whereas the other stimuli are in a non-privileged state. The current study investigated whether the activation states of WM modulate the role of object-based attention in retaining bindings in WM. To address this question, we presented three colored shapes sequentially and added a transparent-motion task (Experiment 1) or a mental rotation task (Experiment 2) into the WM maintenance phase to consume object-based attention. We consistently found that consuming object-based attention led to a larger impairment to bindings relative to constituent features, which is independent of the WM activation states, suggesting that object-based attention is critical in retaining bindings in WM across activation states of WM.

The role of attention in retaining the binding of integral features in working memory.

Previous studies have suggested that retaining bindings in working memory (WM) requires more object-based attention than retaining constituent features. However, we still need to address the object-based attention hypothesis to determine both the generality (Does the object-based attention hypothesis of binding apply to feature bindings other than those tested?) and the reality (Was the observed effect in previous studies an artifact of the testing process?). We addressed these two issues by focusing on the binding of integral features, which was ignored in previous studies. Integral features can be manipulated independently but cannot be attended to or processed independently of each other, and they are primarily perceived in a more unitary fashion. Consequently, integral-feature bindings should be processed as integrated units without the help of extra object-based attention. We examined whether or not the object-based attention hypothesis applied to integral-feature bindings (generality), and these results enabled us to check the reality of the hypothesis. In line with our prediction, we found that a secondary task consuming object-based attention did not selectively impair the binding performance (Experiments 1, 2, 3, 5, and 7). The absence of selective binding impairment was not attributable to the use of an invalid secondary task (Experiment 4), failure to memorize the binding between length and width (Experiment 6), tapping the incorrect type of attention (Experiment 6), the feasibility of feature categorization (Experiment 7), or poor task performance (Experiment 7). Overall, these results suggest that the object-based attention hypothesis does not fit for the integral-feature bindings, and that the pivotal role of object-based attention reported by previous studies was reliable.

Single-cell multiplex chromatin and RNA interactions in aging human brain.

The dynamically organized chromatin complexes often involve multiplex chromatin interactions and sometimes chromatin-associated RNA (caRNA) 1-3. Chromatin complex compositions change during cellular differentiation and aging, and are expected to be highly heterogeneous among terminally differentiated single cells 4-7. Here we introduce the Multi-Nucleic Acid Interaction Mapping in Single Cell (MUSIC) technique for concurrent profiling of multiplex chromatin interactions, gene expression, and RNA-chromatin associations within individual nuclei. Applied to 14 human frontal cortex samples from elderly donors, MUSIC delineates diverse cortical cell types and states. We observed the nuclei exhibiting fewer short-range chromatin interactions are correlated with an "older" transcriptomic signature and with Alzheimer's pathology. Furthermore, the cell type exhibiting chromatin contacts between cis expression quantitative trait loci (cis eQTLs) and a promoter tends to be the cell type where these cis eQTLs specifically affect their target gene's expression. Additionally, the female cortical cells exhibit highly heterogeneous interactions between the XIST non-coding RNA and Chromosome X, along with diverse spatial organizations of the X chromosomes. MUSIC presents a potent tool for exploring chromatin architecture and transcription at cellular resolution in complex tissues.

High throughput direct 3D bioprinting in multiwell plates.

Advances in three dimensional (3D) bioprinting have enabled the fabrication of sophisticated 3D tissue scaffolds for biological and medical applications, where high speed, high throughput production in well plates is a critical need. Here, we present an integrated 3D bioprinting platform based on microscale continuous optical printing, capable of high throughputin siturapid fabrication of complex 3D biomedical samples in multiwell plate formats for subsequent culture and analysis. Our high throughput 3D bioprinter (HT-3DP) was used to showcase constructs of varying spatial geometries of biomimetic significance, tunable mechanical properties, as well as reproducibility. Live hepatocellular carcinoma 3D tissue scaffolds were fabricatedin situin multiwell plates, after which a functional drug response assay against the chemotherapy drug doxorubicin was performed. Dual cell-type populations involving both live hepatocellular carcinoma as well as human umbilical vein endothelial cells were also printed to demonstrate dual-tissue fabrication capability. This work demonstrates a significant advancement in that the production rate of 3D bioprinted tissue scaffolds with controllable spatial architectures and mechanical properties can now be done on a high throughput scale, enabling rapid generation ofin vitro3D tissue models within conventional multiwell cell culture plates for high throughput preclinical drug screening and disease modeling.

Event-based encoding of biological motion and location in visual working memory.

We make use of discrete yet meaningful events to orient ourselves to the dynamic environment. Among these events, biological motion, referring to the movements of animate entities, is one of the most biologically salient. We usually encounter biological motions of multiple human beings taking place simultaneously at distinct locations. How we encode biological motions into visual working memory (VWM) to form a coherent experience of the external world and guide our social behaviour remains unclear. This study for the first time addressed the VWM encoding mechanism of biological motions and their corresponding locations. We tested an event-based encoding hypothesis for biological motion and location: When one element of an event is required to be memorised, the irrelevant element of an event will also be extracted into VWM. We presented participants with three biological motions at different locations and required them to memorise only the biological motions or their locations while ignoring the other dimension. We examined the event-based encoding by probing a distracting effect: If the event-based encoding took place, the change of irrelevant dimension in the probe would lead to a significant distraction and impair the performance of detecting target dimension. We found significant distracting effects, which lasted for 3 s but vanished at 6 s, regardless of the target dimension (biological motions vs. locations, Experiment 1) and the exposure time of memory array (1 s vs. 3 s, Experiment 2). These results together support an event-based encoding mechanism during VWM encoding of biological motions and their corresponding locations.

Three-dimensional bioprinted glioblastoma microenvironments model cellular dependencies and immune interactions.

Brain tumors are dynamic complex ecosystems with multiple cell types. To model the brain tumor microenvironment in a reproducible and scalable system, we developed a rapid three-dimensional (3D) bioprinting method to construct clinically relevant biomimetic tissue models. In recurrent glioblastoma, macrophages/microglia prominently contribute to the tumor mass. To parse the function of macrophages in 3D, we compared the growth of glioblastoma stem cells (GSCs) alone or with astrocytes and neural precursor cells in a hyaluronic acid-rich hydrogel, with or without macrophage. Bioprinted constructs integrating macrophage recapitulate patient-derived transcriptional profiles predictive of patient survival, maintenance of stemness, invasion, and drug resistance. Whole-genome CRISPR screening with bioprinted complex systems identified unique molecular dependencies in GSCs, relative to sphere culture. Multicellular bioprinted models serve as a scalable and physiologic platform to interrogate drug sensitivity, cellular crosstalk, invasion, context-specific functional dependencies, as well as immunologic interactions in a species-matched neural environment.

Rapid 3D bioprinting of decellularized extracellular matrix with regionally varied mechanical properties and biomimetic microarchitecture.

Hepatocellular carcinoma (HCC), as the fifth most common malignant cancer, develops and progresses mostly in a cirrhotic liver where stiff nodules are separated by fibrous bands. Scaffolds that can provide a 3D cirrhotic mechanical environment with complex native composition and biomimetic architecture are necessary for the development of better predictive tissue models. Here, we developed photocrosslinkable liver decellularized extracellular matrix (dECM) and a rapid light-based 3D bioprinting process to pattern liver dECM with tailorable mechanical properties to serve as a platform for HCC progression study. 3D bioprinted liver dECM scaffolds were able to stably recapitulate the clinically relevant mechanical properties of cirrhotic liver tissue. When encapsulated in dECM scaffolds with cirrhotic stiffness, HepG2 cells demonstrated reduced growth along with an upregulation of invasion markers compared to healthy controls. Moreover, an engineered cancer tissue platform possessing tissue-scale organization and distinct regional stiffness enabled the visualization of HepG2 stromal invasion from the nodule with cirrhotic stiffness. This work demonstrates a significant advancement in rapid 3D patterning of complex ECM biomaterials with biomimetic architecture and tunable mechanical properties for in vitro disease modeling.