tRNA anticodon cleavage by target-activated CRISPR-Cas13a effector.

Type VI CRISPR-Cas systems are among the few CRISPR varieties that target exclusively RNA. The CRISPR RNA-guided, sequence-specific binding of target RNAs, such as phage transcripts, activates the type VI effector, Cas13. Once activated, Cas13 causes collateral RNA cleavage, which induces bacterial cell dormancy, thus protecting the host population from the phage spread. We show here that the principal form of collateral RNA degradation elicited by Leptotrichia shahii Cas13a expressed in Escherichia coli cells is the cleavage of anticodons in a subset of transfer RNAs (tRNAs) with uridine-rich anticodons. This tRNA cleavage is accompanied by inhibition of protein synthesis, thus providing defense from the phages. In addition, Cas13a-mediated tRNA cleavage indirectly activates the RNases of bacterial toxin-antitoxin modules cleaving messenger RNA, which could provide a backup defense. The mechanism of Cas13a-induced antiphage defense resembles that of bacterial anticodon nucleases, which is compatible with the hypothesis that type VI effectors evolved from an abortive infection module encompassing an anticodon nuclease.

Combinatorial Microgels for 3D ECM Screening and Heterogeneous Microenvironmental Culture of Primary Human Hepatic Stellate Cells.

Nonalcoholic fatty liver disease affects 30% of the United States population and its progression can lead to nonalcoholic steatohepatitis (NASH), and increased risks for cirrhosis and hepatocellular carcinoma. NASH is characterized by a highly heterogeneous liver microenvironment created by the fibrotic activity of hepatic stellate cells (HSCs). While HSCs have been widely studied in 2D, further advancements in physiologically relevant 3D culture platforms for the in vitro modeling of these heterogeneous environments are needed. In this study, the use of stiffness-variable, extracellular matrix (ECM) protein-conjugated polyethylene glycol microgels as 3D cell culture scaffolds to modulate HSC activation is demonstrated. These microgels as a high throughput ECM screening system to identify HSC matrix remodeling and metabolic activities in distinct heterogeneous microenvironmental conditions are further employed. The 6 kPa fibronectin microgels are shown to significantly increase HSC matrix remodeling and metabolic activities in single or multiple-component microenvironments. Overall, heterogeneous microenvironments consisting of multiple distinct ECM microgels promoted a decrease in HSC matrix remodeling and metabolic activities compared to homogeneous microenvironments. The study envisions this ECM screening platform being adapted to a broad number of cell types to aid the identification of ECM microenvironments that best recapitulate the desired phenotype, differentiation, or drug efficacy.

Cell Chirality of Micropatterned Endometrial Microvascular Endothelial Cells.

Chirality is an intrinsic cellular property that describes cell polarization biases along the left-right axis, apicobasal axis, or front-rear axes. Cell chirality plays a significant role in the arrangement of organs in the body as well as in the orientation of organelles, cytoskeletons, and cells. Vascular networks within the endometrium, the mucosal inner lining of the uterus, commonly display spiral architectures that rapidly form across the menstrual cycle. Herein, the role of endometrial-relevant extracellular matrix stiffness, composition, and soluble signals on endometrial endothelial cell chirality is systematically examined using a high-throughput microarray. Endometrial endothelial cells display marked patterns of chirality as individual cells and as cohorts in response to substrate stiffness and environmental cues. Vascular networks formed from endometrial endothelial cells also display shifts in chirality as a function of exogenous hormones. Changes in cellular-scale chirality correlate with changes in vascular network parameters, suggesting a critical role for cellular chirality in directing endometrial vessel network organization.

Cell Chirality of Micropatterned Endometrial Microvascular Endothelial Cells.

Chirality is an intrinsic cellular property that describes cell polarization biases along the left-right axis, apicobasal axis, or front-rear axes. Cell chirality plays a significant role in the arrangement of organs in the body as well as the orientation of organelles, cytoskeletons, and cells. Vascular networks within the endometrium, the mucosal inner lining of the uterus, commonly display spiral architectures that rapidly form across the menstrual cycle. Herein, we systematically examine the role of endometrial-relevant extracellular matrix stiffness, composition, and soluble signals on endometrial endothelial cell chirality using a high-throughput microarray. Endometrial endothelial cells display marked patterns of chirality as individual cells and as cohorts in response to substrate stiffness and environmental cues. Vascular networks formed from endometrial endothelial cells also display shifts in chirality as a function of exogenous hormones. Changes in cellular-scale chirality correlate with changes in vascular network parameters, suggesting a critical role for cellular chirality in directing endometrial vessel network organization.

Combinatorial extracellular matrix cues with mechanical strain induce differential effects on myogenesis in vitro.

Skeletal muscle regeneration remains a clinical unmet need for volumetric muscle loss and atrophy where muscle function cannot be restored to prior capacity. Current experimental approaches do not account for the complex microenvironmental factors that modulate myogenesis. In this study we developed a biomimetic tissue chip platform to systematically study the combined effects of the extracellular matrix (ECM) microenvironment and mechanical strain on myogenesis of murine myoblasts. Using stretchable tissue chips composed of collagen I (C), fibronectin (F) and laminin (L), as well as their combinations thereof, we tested the addition of mechanical strain regimens on myogenesis at the transcriptomic and translational levels. Our results show that ECMs have a significant effect on myotube formation in C2C12 murine myoblasts. Under static conditions, laminin substrates induced the longest myotubes, whereas fibronectin produced the widest myotubes. Combinatorial ECMs showed non-intuitive effects on myotube formation. Genome-wide analysis revealed the upregulation in actin cytoskeletal related genes that are suggestive of myogenesis. When mechanical strain was introduced to C + F + L combinatorial ECM substrates in the form of constant or intermittent uniaxial strain at low (5%) and high (15%) levels, we observed synergistic enhancements in myotube width, along with transcriptomic upregulation in myosin heavy chain genes. Together, these studies highlight the complex role of microenvironmental factors such as ECM interactions and strain on myotube formation and the underlying signaling pathways.

Intravenous Fluid Resuscitation Capabilities in Simulated Reduced Gravity.

BACKGROUND: Critical care for exploration space missions may require intravenous (IV) fluid resuscitation therapy. Resource constraints may limit availability of standard, Earth-based infusion technologies. The effect of variable acceleration on infusion flow rates using simple fluid resuscitation supplies was investigated.METHODS: Infusions of water or blood analog (40% glycerol) from a 1 L IV bag were performed using pressure bag augmentation at 0, 150, or 300 mmHg. The solution bag rested on an adjustable mount, configured to different heights to simulate relevant gravitational accelerations (1 G, Martian G, lunar G, and 0 G). The bag emptied through an IV line with a 14- or 20-gauge angiocath into a 3-mmHg venous pressure reservoir. Flow rates were measured using an in-line flow probe. Three determinations were made for each test condition.RESULTS: Temporal flow rate data for all test conditions displayed one-phase exponential decay. At 300 mmHg pressurization, maximum infusion rates ranged from 92-222 mL ⋅ min-1 for water and from 21-49 mL ⋅ min-1 for blood analog. All reduced gravity conditions had significantly longer infusion times in comparison to 1 G for both test solutions.DISCUSSION: Reduced acceleration significantly altered flow rates and infusion times for fluid resuscitation. Fluid resuscitation protocols specify a desired volume to infuse for a target time (e.g., 20-30 mL ⋅ min-1 for a 75-kg adult). This data demonstrates that this protocol parameter can be achieved with infusion pressure bag augmentation alone and provides information for the refinement of fluid resuscitation protocols for exploration space missions.Pantalos GM, Heidel JS, Jain IM, Warner SE, Barefoot TL, Baker RO, Hailey M. Intravenous fluid resuscitation capabilities in simulated reduced gravity. Aerosp Med Hum Perform. 2023; 94(8):596-603.

Effect of distinct ECM microenvironments on the genome-wide chromatin accessibility and gene expression responses of hepatic stellate cells.

Hepatic stellate cells (HSCs) are one of the primary drivers of liver fibrosis in non-alcoholic fatty liver disease. Although HSC activation in liver disease is associated with changes in extracellular matrix (ECM) deposition and remodeling, it remains unclear how ECM regulates the phenotypic state transitions of HSCs. Using high-throughput cellular microarrays, coupled with genome-wide ATAC and RNA sequencing within engineered ECM microenvironments, we investigated the effect of ECM and substrate stiffness on chromatin accessibility and resulting gene expression in activated primary human HSCs. Cell microarrays demonstrated the cooperative effects of stiffness and ECM composition on H3K4 and H3K9 methylation/acetylation. ATAC sequencing revealed higher chromatin accessibility in HSCs on 1kPa compared to 25kPa substrates for all ECM conditions. Gene set enrichment analysis using RNA sequencing data of HSCs in defined ECM microenvironments demonstrated higher enrichment of NAFLD and fibrosis-related genes in pre-activated HSCs on 1kPa relative to 25kPa. Overall, these findings are indicative of a microenvironmental adaptation response in HSCs, and the acquisition of a persistent activation state. Combined ATAC/RNA sequencing analyses enabled identification of candidate regulatory factors, including HSD11B1 and CEBPb. siRNA-mediated knockdown of HSD11b1 and CEBPb demonstrated microenvironmental controlled reduction in fibrogenic markers in HSCs. STATEMENT OF SIGNIFICANCE: Hepatic stellate cells (HSCs) are one of the primary drivers of liver fibrosis in non-alcoholic fatty liver disease. Although HSC activation in liver disease is associated with changes in extracellular matrix (ECM) deposition and remodeling, it remains unclear how ECM regulates the phenotypic state transitions of HSCs. Using high-throughput cellular microarrays, coupled with genome-wide ATAC and RNA sequencing within engineered ECM microenvironments, we investigated the effect of ECM and substrate stiffness on chromatin accessibility and resulting gene expression in activated primary human HSCs. Overall, these findings were indicative of a microenvironmental adaptation response in HSCs, and the acquisition of a persistent activation state. Combined ATAC/RNA sequencing analyses enabled identification of candidate regulatory factors, including HSD11B1 and CEBPb. siRNA-mediated knockdown of HSD11b1 and CEBPb demonstrated microenvironmental controlled reduction in fibrogenic markers in HSCs.

Delineating cooperative effects of Notch and biomechanical signals on patterned liver differentiation.

Controlled in vitro multicellular culture systems with defined biophysical microenvironment have been used to elucidate the role of Notch signaling in the spatiotemporal regulation of stem and progenitor cell differentiation. In addition, computational models incorporating features of Notch ligand-receptor interactions have provided important insights into Notch pathway signaling dynamics. However, the mechanistic relationship between Notch-mediated intercellular signaling and cooperative microenvironmental cues is less clear. Here, liver progenitor cell differentiation patterning was used as a model to systematically evaluate the complex interplay of cellular mechanics and Notch signaling along with identifying combinatorial mechanisms guiding progenitor fate. We present an integrated approach that pairs a computational intercellular signaling model with defined microscale culture configurations provided within a cell microarray platform. Specifically, the cell microarray-based experiments were used to validate and optimize parameters of the intercellular Notch signaling model. This model incorporated the experimentally established multicellular dimensions of the cellular microarray domains, mechanical stress-related activation parameters, and distinct Notch receptor-ligand interactions based on the roles of the Notch ligands Jagged-1 and Delta-like-1. Overall, these studies demonstrate the spatial control of mechanotransduction-associated components, key growth factor and Notch signaling interactions, and point towards a possible role of E-Cadherin in translating intercellular mechanical gradients to downstream Notch signaling.

Role of Extracellular Matrix Biomolecules on Endometrial Epithelial Cell Attachment and Cytokeratin 18 Expression on Gelatin Hydrogels.

The endometrium undergoes profound changes in tissue architecture and composition, both during the menstrual cycle as well as in the context of pregnancy. Dynamic remodeling processes of the endometrial extracellular matrix (ECM) are a major element of endometrial homeostasis, including changes across the menstrual cycle. A critical element of this tissue microenvironment is the endometrial basement membrane, a specialized layer of proteins that separates the endometrial epithelium from the underlying endometrial ECM. Bioengineering models of the endometrial microenvironment that present an appropriate endometrial ECM and basement membrane may provide an improved environment to study endometrial epithelial cell (EEC) function. Here, we exploit a tiered approach using two-dimensional high-throughput microarrays and three-dimensional gelatin hydrogels to define patterns of EEC attachment and cytokeratin 18 (CK18) expression in response to combinations of endometrial basement membrane proteins. We identify combinations (collagen IV + tenascin C; collagen I + collagen III; hyaluronic acid + tenascin C; collagen V; collagen V + hyaluronic acid; collagen III; and collagen I) that facilitate increased EEC attachment, increased CK18 intensity, or both. We also identify significant EEC mediated remodeling of the methacrylamide-functionalized gelatin matrix environment via analysis of nascent protein deposition. Together, we report efforts to tailor the localization of basement membrane-associated proteins and proteoglycans in order to investigate tissue-engineered models of the endometrial microenvironment.

Simulated confluence on micropatterned substrates correlates responses regulating cellular differentiation.

While cells are known to behave differently based on the size of micropatterned islands, and this behavior is thought to be related to cell size and cell-cell contacts, the exact threshold for this difference between small and large islands is unknown. Furthermore, while cell size and cell-cell contacts can be easily manipulated on small islands, they are harder to measure and continually monitor on larger islands. To investigate this size threshold, and to explore cell size, cell-cell contacts, and differentiation, we use a previously established simulation to plan experiments and explain results that we could not explain from experiments alone. We use five seeding densities covering three orders of magnitude over 25-500 µm diameter islands to examine markers of proliferation and differentiation in bone marrow-derived mesenchymal cells (cell line). We show that osteogenic markers are most accurately described as a function of confluence for larger islands, but a function of time for smaller islands. We further show, using results of the simulation, that cell size and cell-cell contacts are also related to confluence on larger islands, but only cell-cell contacts are related to confluence on small islands. This study uses simulations to explain experimental results that could not be explained from experiments alone. Together, the simulations and experiments in this study show different differentiation patterns on large and small islands, and this simulation may be useful in planning future studies related to this study.

High throughput interrogation of human liver stellate cells reveals microenvironmental regulation of phenotype.

Liver fibrosis is a common feature of progressive liver disease and is manifested as a dynamic series of alterations in both the biochemical and biophysical properties of the liver. Hepatic stellate cells (HSCs) reside within the perisinusoidal space of the liver sinusoid and are one of the main drivers of liver fibrosis, yet it remains unclear how changes to the sinusoidal microenvironment impact HSC phenotype in the context of liver fibrosis. Cellular microarrays were used to examine and deconstruct the impacts of bio-chemo-mechanical changes on activated HSCs in vitro. Extracellular matrix (ECM) composition and stiffness were found to act individually and in combination to regulate HSC fibrogenic phenotype and proliferation. Hyaluronic acid and collagen III promoted elevated collagen I expression while collagen IV mediated a decrease. Previously activated HSCs exhibited reduced lysyl oxidase (Lox) expression as array substrate stiffness increased, with less dependence on ECM composition. Collagens III and IV increased HSC proliferation, whereas hyaluronic acid had the opposite effect. Meta-analysis performed on these data revealed distinct phenotypic clusters (e.g. low fibrogenesis/high proliferation) as a direct function of their microenvironmental composition. Notably, soft microenvironments mimicking healthy tissue (1 kPa), promoted higher levels of intracellular collagen I and Lox expression in activated HSCs, compared to stiff microenvironments mimicking fibrotic tissue (25 kPa). Collectively, these data suggest potential HSC functional adaptations in response to specific bio-chemo-mechanical changes relevant towards the development of therapeutic interventions. These findings also underscore the importance of the microenvironment when interrogating HSC behavior in healthy, disease, and treatment settings. STATEMENT OF SIGNIFICANCE: In this work we utilized high-throughput cellular microarray technology to systematically interrogate the complex interactions between HSCs and their microenvironment in the context of liver fibrosis. We observed that HSC phenotype is regulated by ECM composition and stiffness, and that these phenotypes can be classified into distinct clusters based on their microenvironmental context. Moreover, the range of these phenotypic responses to microenvironmental stimuli is substantial and a direct consequence of the combinatorial pairing of ECM protein and stiffness signals. We also observed a novel role for microenvironmental context in affecting HSC responses to potential fibrosis therapeutics.

Defining the seed sequence of the Cas12b CRISPR-Cas effector complex.

Target binding by CRISPR-Cas ribonucleoprotein effectors is initiated by the recognition of double-stranded PAM motifs by the Cas protein moiety followed by destabilization, localized melting, and interrogation of the target by the guide part of CRISPR RNA moiety. The latter process depends on seed sequences, parts of the target that must be strictly complementary to CRISPR RNA guide. Mismatches between the target and CRISPR RNA guide outside the seed have minor effects on target binding, thus contributing to off-target activity of CRISPR-Cas effectors. Here, we define the seed sequence of the Type V Cas12b effector from Bacillus thermoamylovorans. While the Cas12b seed is just five bases long, in contrast to all other effectors characterized to date, the nucleotide base at the site of target cleavage makes a very strong contribution to target binding. The generality of this additional requirement was confirmed during analysis of target recognition by Cas12b effector from Alicyclobacillus acidoterrestris. Thus, while the short seed may contribute to Cas12b promiscuity, the additional specificity determinant at the site of cleavage may have a compensatory effect making Cas12b suitable for specialized genome editing applications.

Spatial patterning of liver progenitor cell differentiation mediated by cellular contractility and Notch signaling.

The progenitor cells of the developing liver can differentiate toward both hepatocyte and biliary cell fates. In addition to the established roles of TGFß and Notch signaling in this fate specification process, there is increasing evidence that liver progenitors are sensitive to mechanical cues. Here, we utilized microarrayed patterns to provide a controlled biochemical and biomechanical microenvironment for mouse liver progenitor cell differentiation. In these defined circular geometries, we observed biliary differentiation at the periphery and hepatocytic differentiation in the center. Parallel measurements obtained by traction force microscopy showed substantial stresses at the periphery, coincident with maximal biliary differentiation. We investigated the impact of downstream signaling, showing that peripheral biliary differentiation is dependent not only on Notch and TGFß but also E-cadherin, myosin-mediated cell contractility, and ERK. We have therefore identified distinct combinations of microenvironmental cues which guide fate specification of mouse liver progenitors toward both hepatocyte and biliary fates.

A Review on Biomechanics of Anterior Cruciate Ligament and Materials for Reconstruction.

The anterior cruciate ligament is one of the six ligaments in the human knee joint that provides stability during articulations. It is relatively prone to acute and chronic injuries as compared to other ligaments. Repair and self-healing of an injured anterior cruciate ligament are time-consuming processes. For personnel resuming an active sports life, surgical repair or replacement is essential. Untreated anterior cruciate ligament tear results frequently in osteoarthritis. Therefore, understanding of the biomechanics of injury and properties of the native ligament is crucial. An abridged summary of the prominent literature with a focus on key topics on kinematics and kinetics of the knee joint and various loads acting on the anterior cruciate ligament as a function of flexion angle is presented here with an emphasis on the gaps. Briefly, we also review mechanical characterization composition and anatomy of the anterior cruciate ligament as well as graft materials used for replacement/reconstruction surgeries. The key conclusions of this review are as follows: (a) the highest shear forces on the anterior cruciate ligament occur during hyperextension/low flexion angles of the knee joint; (b) the characterization of the anterior cruciate ligament at variable strain rates is critical to model a viscoelastic behavior; however, studies on human anterior cruciate ligament on variable strain rates are yet to be reported; (c) a significant disparity on maximum stress/strain pattern of the anterior cruciate ligament was observed in the earlier works; (d) nearly all synthetic grafts have been recalled from the market; and (e) bridge-enhanced repair developed by Murray is a promising technique for anterior cruciate ligament reconstruction, currently in clinical trials. It is important to note that full extension of the knee is not feasible in the case of most animals and hence the loading pattern of human ACL is different from animal models. Many of the published reviews on the ACL focus largely on animal ACL than human ACL. Further, this review article summarizes the issues with autografts and synthetic grafts used so far. Autografts (patellar tendon and hamstring tendon) remains the gold standard as nearly all synthetic grafts introduced for clinical use have been withdrawn from the market. The mechanical strength during the ligamentization of autografts is also highlighted in this work.

Altered stoichiometry Escherichia coli Cascade complexes with shortened CRISPR RNA spacers are capable of interference and primed adaptation.

The Escherichia coli type I-E CRISPR-Cas system Cascade effector is a multisubunit complex that binds CRISPR RNA (crRNA). Through its 32-nucleotide spacer sequence, Cascade-bound crRNA recognizes protospacers in foreign DNA, causing its destruction during CRISPR interference or acquisition of additional spacers in CRISPR array during primed CRISPR adaptation. Within Cascade, the crRNA spacer interacts with a hexamer of Cas7 subunits. We show that crRNAs with a spacer length reduced to 14 nucleotides cause primed adaptation, while crRNAs with spacer lengths of more than 20 nucleotides cause both primed adaptation and target interference in vivo Shortened crRNAs assemble into altered-stoichiometry Cascade effector complexes containing less than the normal amount of Cas7 subunits. The results show that Cascade assembly is driven by crRNA and suggest that multisubunit type I CRISPR effectors may have evolved from much simpler ancestral complexes.