Explore the vast range of titles available.

The progenitor cells of the developing liver can differentiate toward both hepatocyte and biliary cell fates. In addition to the established roles of TGFβ 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 TGFβ 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. Children are said to be a product of both nature and nurture – of their genes and the environment in which they are raised. The cells of the growing liver are not so different in this sense. As the liver of a fetus develops, immature cells called liver progenitors mature to become one of two types of adult cells: the hepatocytes that form the bulk of the liver, or the biliary cells that make up the bile duct. The traditional view is that genetic factors mainly control which cell type the progenitor cells become. However, recent research suggests that the environment around the cells matters more in this process than once thought. Cells can respond to the physical properties of their environment, such as the structure and stiffness of the surrounding tissue. These properties change as the liver develops, and can also be altered by disease. For example, damaged liver cells can spit out proteins that harden and form stiff scars. This raises a question: do changes in stiffness affect how progenitor cells behave? To answer this question, Kaylan et al. printed collagen in circular patterns and grew liver progenitor cells on them. The cells at the edges of the circular patterns matured into bile duct cells, while those in the center became hepatocytes. The stiffness felt by the cells was then determined by measuring the level of mechanical stress that they experienced. This revealed that the cells at the edge of the collagen pattern – the cells that became bile duct cells – were under most stress. In addition, more bile duct cells formed when progenitor cells were grown on a stiffer collagen pattern. Overall, the results reported by Kaylan et al. suggest that the stiffness of the environment, and the resulting stresses on a progenitor cell, can influence how it matures. As well as helping us to understand how the liver develops, this knowledge could also help us to treat a group of diseases called cholangiopathies, in which the bile ducts become inflamed. These diseases are thought to be caused by certain cells (which are similar to liver progenitor cells) maturing to become incorrect cell types. Future studies could determine if preventing changes in stiffness in the environment of these cells, or slowing their response to such changes, would help patients.

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.

The gender gap in drug use is narrowing in regions where access to criminalized substances, such as opioids, is increasing. While research shows that substance use is gendered, less is known about the cultural norms and values shaping women’s drug use, as most studies focus on men. Cross-national comparisons of cultural models of addiction are needed to better understand how addiction is perceived and to inform culturally responsive treatment approaches for women. This study examined cultural models of addiction among reproductive-aged women receiving treatment for substance misuse in London, Toronto, and Delhi. Participants completed a semi-structured questionnaire with open-ended and free-list prompts. Findings revealed shared cultural models attributing drug use to psychological factors, such as self-medicating to manage negative emotions or enhance positive ones, as well as relational, developmental, and biological influences. In conclusion, the study highlights the importance of incorporating cultural models into research and treatment. By using an inductive approach to explore meanings surrounding drug use among people in recovery, researchers can better understand how interventions are received and interpreted through existing internal frameworks.

Ultraviolet A and B (UVA 320–400 nm and UVB 280–320 nm) induced cyclobutane-pyrimidine dimers (CPDs) are the most critical lesions caused by environmental sun exposure. Here we show that CPD removal is accelerated when, in addition to UV, cells are simultaneously exposed to water-filtered near-infrared (nIR, 750–1600 nm). The described effect is dose-dependent on the nIR-dose and is found in skin keratinocytes and fibroblasts. Accelerated removal of CPDs, which coincides with chromatin relaxation and faster CPD recognition, occurs after nIR exposure. While nIR alone does not affect cellular survival, co-exposure to UVB leads to reduced cellular survival and an increased number of mutations. Increasing single strand break levels (SSB) occur transiently after nIR exposure and independent of reactive oxygen species (ROS) formation. These data suggest that the rate-limiting step in the NER repair process – damage recognition – is facilitated by nIR-induced chromatin relaxation, causing the accumulation of unnatural high levels of SSBs and single stranded DNA, unfavourable for the cell fate resulting in reduced survival and increased mutation rates. Since nIR modulates the UV-dependent damage response, risk estimation of solar radiation-induced DNA damage should not only consider the UV components but also include the nIR fraction of the solar spectrum.

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. The development and utility of stiffness‐variable, ECM protein‐conjugated polyethylene glycol microgels as 3D cell culture scaffolds to modulate human hepatic stellate cell activation is demonstrated. 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.

Despite the therapeutic potential of mesenchymal stromal cells (MSC), there is limited understanding of optimal extracellular matrix (ECM) environments to manufacture these cells. We developed tissue chips to study the effects of multi-factorial ECM environments under manufacturable stiffness ranges and multi-component ECM compositions. Manufacturing qualities of cell expansion potential, immunomodulation, and differentiation capacity were examined. The results show stiffness effects, with 900 kPa substrates supporting higher proliferation and osteogenic differentiation, along with anti-inflammatory IL-10 expression, whereas 150 kPa substrates promoted adipogenic differentiation at 150 kPa, suggesting that optimal ECM environments may differ based on manufacturing goals. ECM biochemistries containing fibronectin and laminin further modulated MSC manufacturing qualities across various stiffnesses. Proteomic and transcriptomic analyses revealed unique ECM combinations that induced higher levels of angiogenic and immunomodulatory cytokines, compared to single factor ECMs. These findings demonstrate that optimized ECM environments enhance MSC manufacturing quality.

Volumetric muscle loss (VML) is characterized by permanent tissue impairment resulting from critically-sized muscle loss. We performed time-series transcriptomic and proteomic analyses to reveal key mediators of irreversible pathological remodeling after induction of VML in mice. The dynamics of gene and protein expression patterns were analyzed for up to 3 weeks after muscle injury. RNA Sequencing revealed transcriptional patterns that show rapid upregulation or downregulation shortly after injury, among which a subset of genes failed to return to pre-injury levels within 3 weeks after VML. Time-series analysis revealed gene clusters with sustained upregulation after 3 weeks, including those associated with extracellular matrix remodeling and inflammation, whereas the gene clusters having sustained downregulation were associated with mitochondrial function and metabolism. We further identified and as novel molecular mediators of the pathological remodeling process. This work demonstrates the utility of time-series analysis to reveal dysregulated pathways in the setting of VML.

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. Integrating cellular microarrays, experimental and computational data for liver progenitor cell differentiation patterning, the interplay between Notch signaling and cellular mechanics is assessed.

Bacteria and phages are constantly battling an evolutionary arms race where they developed several defense strategies to outcompete each other. Prokaryotes utilize CRISPR-Cas systems, an adaptive immune mechanism to protect themselves by targeting phage genomes in a sequence specific manner. Type VI systems are a unique subtype of Class 2 CRISPR-Cas systems which provide host immunity by exclusively targeting RNA. The Type VI nuclease Cas13, gets activated upon target RNA recognition and causes ‘collateral’ RNA cleavage in trans, which leads to bacterial cell dormancy and protection of the host cells. However, due to a lack of in vivo studies, the mechanistic details of the RNA-targeting activity of Type VI systems remains unclear and raises questions about its role as a prokaryotic defense system.In this work, we conducted in vivo and in vitro analyses to show that Cas13a effector from Leptotrichia shahii (Lsh) is a specific RNA-targeting nuclease which does not exhibit promiscuous indiscriminate RNase activity as previously suggested. We show that tRNA anticodon loops are true cleavage substrates for target-activated LshCas13a and these cleavages are preferentially made in uridine rich residues.On the other hand, spacer acquisition is an important process in making CRISPR-Cas systems ‘adaptive’ in nature, where the CRISPR array can store memories of past phage infections and pass onto bacterial progeny. Since the Type VI-A system targets RNA and has no known associated reverse transcriptases, it is important to understand how this system acquires new spacers.Here, we developed an oligo adaptation assay to test spacer integration in the Type VI-A CRISPR array. We showed that RNA-targeting LshCas13a systems can acquire double-stranded DNA oligos as spacers in the CRISPR array utilizing the Cas1 and Cas2 adaptation proteins. We also demonstrated that RNA oligos are not acquired by the Lsh Type VI-A CRISPR systems.In light of these results, we also presented a model of Type VI-A CRISPR-Cas adaptive immunity to describe its function as an adaptive prokaryotic defense system.

Liver disease is a major public health concern, and one of the key sources of tissue and organ dysregulation in liver disease is the onset of liver fibrosis. Liver has an immense capacity to repair and regenerate, which is also impaired in later stages of liver fibrosis. Over the past decade, the demand for liver transplant has been increasing. There are multiple chronic diseases that lead to liver failure, and subsequent loss of regeneration capacity. The major proportion of candidates listed on the waiting list for transplant have been diagnosed with either Alcoholic Liver disease or liver disease due non-alcoholic fatty liver disease (NAFLD). This proportion has also been increasing over the past decade, due to the higher prevalence of diabetes and obesity. This thesis focuses on developing tools and studies to meet and/or reduce this increasing clinical need for liver transplants. I have developed in vitro platforms to study the microenvironmental control of disease phenotype and basic developmental processes. The objective of the study was to examine how combinations of various microenvironmental factors such the extracellular matrix, substrate stiffness and cellular forces affect cellular behavior in the context of liver fibrosis and liver development. Liver fibrosis produces changes in the composition of the liver and has profound repercussions for cell behavior and function, especially in tissue microenvironments. The makeup and configuration of the microenvironment is critical in directing cell behaviors due to the myriad extracellular cues it presents. Cellular microarray technology is a powerful platform for efficiently and robustly dissecting the effects of multiple microenvironmental parameters simultaneously. The basic understanding of the complex behavior of liver cells and delineating the various factors affecting their behavior is crucial to design better interventions for chronic liver disease and ultimately developing artificial livers for transplants.