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.

Benefits of Robot-Assisted Upper-Limb Rehabilitation from the Subacute Stage after a Stroke of Varying Severity: A Multicenter Randomized Controlled Trial.

BACKGROUND: The aim of this study was to compare the clinical effectiveness of robot-assisted therapy with that of conventional occupational therapy according to the onset and severity of stroke. METHODS: In this multicenter randomized controlled trial, stroke patients were randomized (1:1) to receive robot-assisted therapy or conventional occupational therapy. The robot-assisted training group received 30 min of robot-assisted therapy twice and 30 min of conventional occupational therapy daily, while the conventional therapy group received 90 min of occupational therapy. Therapy was conducted 5 days/week for 4 weeks. The primary outcome was the Wolf Motor Function Test (WMFT) score after 4 and 8 weeks of therapy. RESULTS: Overall, 113 and 115 patients received robot-assisted and conventional therapy, respectively. The WMFT score after robot-assisted therapy was not significantly better than that after conventional therapy, but there were significant improvements in the Motricity Index (trunk) and the Fugl-Meyer Assessment. After robot-assisted therapy, wrist strength significantly improved in the subacute or moderate-severity group of stroke patients. CONCLUSIONS: Robot-assisted therapy improved the upper-limb functions and activities of daily living (ADL) performance as much as conventional occupational therapy. In particular, it showed signs of more therapeutic effectiveness in the subacute stage or moderate-severity group.

Regulatory role of cholesterol in modulating actin dynamics and cell adhesive interactions in the trabecular meshwork.

The trabecular meshwork (TM) tissue plays a crucial role in maintaining intraocular pressure (IOP) homeostasis. Increased TM contractility and stiffness are directly correlated with elevated IOP. Although cholesterol is known to be a determinant of glaucoma occurrence and elevated IOP, the underlying mechanisms remain elusive. In this study, we used human TM (HTM) cells to unravel the effects of cholesterol on TM stiffness. We achieved this by performing acute cholesterol depletion with Methyl-ß-cyclodextrin (MßCD) and cholesterol enrichment/replenishment with MßCD cholesterol complex (CHOL). Interestingly, cholesterol depletion triggered notable actin depolymerization and decreased focal adhesion formation, while enrichment/replenishment promoted actin polymerization, requiring the presence of actin monomers. Using a specific reporter of phosphatidylinositol 4,5-bisphosphate (PIP2), we demonstrated that cholesterol depletion decreases PIP2 levels on the cell membrane, whereas enrichment increases them. Given the critical role of PIP2 in actin remodeling and focal adhesion formation, we postulate that cholesterol regulates actin dynamics by modulating PIP2 levels on the membrane. Furthermore, we showed that cholesterol levels regulate integrin α5ß1 and αVß3 distribution and activation, subsequently altering cell-extracellular matrix (ECM) interactions. Notably, the depletion of cholesterol, as a major lipid constituent of the cell membrane, led to a decrease in HTM cell membrane tension, which was reversed upon cholesterol replenishment. Overall, our systematic exploration of cholesterol modulation on TM stiffness highlights the critical importance of maintaining appropriate membrane and cellular cholesterol levels for achieving IOP homeostasis.

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

Non-alcoholic fatty liver disease affects 30% of the United States population and its progression can lead to non-alcoholic steatohepatitis (NASH), which can result in 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, we have demonstrated the use of stiffness-variable, ECM protein-conjugated polyethylene glycol microgels as 3D cell culture scaffolds to modulate HSC activation. We further employed these microgels as a high throughput ECM screening system to identify HSC matrix remodeling and metabolic activities in distinct heterogeneous microenvironmental conditions. In particular, 6 kPa fibronectin microgels were 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. We envision 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.

Spatially Defined Cell-Secreted Protein Detection Using Granular Hydrogels: µGeLISA.

Intercellular communication through secreted proteins is necessary in essential processes such as embryo and limb development, disease progression, and immune responses. There exist many techniques to study bulk solution protein concentrations, but there is a limited set of tools to study the concentrations of cell-secreted proteins in situ within diverse cell platforms while retaining spatial information. In this study, we have developed a microgel system that is able to quantitatively measure the cell-secreted protein concentration within defined three-dimensional culture configurations with single-cell spatial resolution, called µGeLISA (microgel-linked immunosorbent assay). This system, which is based on the surface modification of polyethylene glycol microgels, was able to detect interleukin 6 (IL-6) concentrations of 2.21-21.86 ng/mL. Microgels were also able to detect cell spheroid-secreted IL-6 and distinguish between low- and high-secreting single cells. The system was also adapted to measure the concentration of cell-secreted matrix metalloproteinase-2 (MMP-2). µGeLISA represents a highly versatile system with a straightforward fabrication process that can be adapted toward the detection of secreted proteins within a diverse range of cell culture configurations.

Can a Biomechanical Foot Orthosis Affect Gait in Patients With Hallux Valgus? A Pilot Study.

OBJECTIVE: To investigate the effects of customized biomechanical foot orthosis (BFO) on kinematic data during gait in patients with hallux valgus (HV) deformities and compare the results with those of a normal control group. METHODS: Ten patients with HV deformities and 10 healthy volunteers were enrolled in this study. HV deformity was diagnosed using biomechanical and radiological assessments by a rehabilitation physician. Patients received the customized BFO manufactured at a commercial orthosis laboratory (Biomechanics, Goyang, South Korea) according to the strictly defined procedure by a single experienced technician. The spatiotemporal and kinematic data acquired by the Vicon 3D motion capture system (Oxford Metrics, Oxford, UK) were compared between the intervention groups (control vs. HV without orthosis) and between the HV groups (with vs. without orthosis). RESULTS: The temporal-spatial and kinematic parameters of the HV group were significantly different from those of the control group. After applying BFO to the HV group, significantly increased ranges of plantar flexion motion and hindfoot inversion were observed. Furthermore, the HV group with BFO showed improved gait cadence, walking speed, and stride length, although the results were not statistically significant. CONCLUSION: Our results suggest that it is imperative to understand the pathophysiology of HV, and the application of customized BFO can be useful for improving kinematics in HV deformities.

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.

Integration of Hydrogel Microparticles With Three-Dimensional Liver Progenitor Cell Spheroids.

The study of the liver progenitor cell microenvironment has demonstrated the important roles of both biochemical and biomechanical signals in regulating the progenitor cell functions that underlie liver morphogenesis and regeneration. While controllable two-dimensional in vitro culture systems have provided key insights into the effects of growth factors and extracellular matrix composition and mechanics on liver differentiation, it remains unclear how microenvironmental signals may differentially affect liver progenitor cell responses in a three-dimensional (3D) culture context. In addition, there have only been limited efforts to engineer 3D culture models of liver progenitor cells through the tunable presentation of microenvironmental stimuli. We present an in vitro model of 3D liver progenitor spheroidal cultures with integrated polyethylene glycol hydrogel microparticles for the internal presentation of modular microenvironmental cues and the examination of the combinatorial effects with an exogenous soluble factor. In particular, treatment with the growth factor TGFß1 directs differentiation of the spheroidal liver progenitor cells toward a biliary phenotype, a behavior which is further enhanced in the presence of hydrogel microparticles. We further demonstrate that surface modification of the hydrogel microparticles with heparin influences the behavior of liver progenitor cells toward biliary differentiation. Taken together, this liver progenitor cell culture system represents an approach for controlling the presentation of microenvironmental cues internalized within 3D spheroidal aggregate cultures. Overall, this strategy could be applied toward the engineering of instructive microenvironments that control stem and progenitor cell differentiation within a 3D context for studies in tissue engineering, drug testing, and cellular metabolism.

Publicly Available Internet Content as a HIV/STI Prevention Intervention for Urban Youth.

Sexual and racial minority adolescents and young adults account for the most substantial number of new HIV infections in the United States. Numerous publicly available websites and YouTube videos contain HIV/STI prevention information that is culturally tailored to racial and ethnic minorities, and gay and bisexual youth. However, the effect of this easily accessible Internet content on adolescent and young adult HIV/STI related knowledge, attitudes and behaviors is unknown. We assembled a HIV/STI Internet intervention from publicly available online sources, including YouTube and privately and publicly hosted websites. We tested the preliminary efficacy of this internet intervention by means of a randomized controlled pilot study with 60 diverse adolescents and young adults recruited in Providence, RI (mean age 18.6 years, 62% male, 52% Black/African American, 36% Hispanic, 47% non-heterosexual). Youth who received links to publicly accessible online prevention content by email had a significant improvement in HIV self-efficacy (p < .05) and a significant reduction in unprotected vaginal or anal sex (12.5 vs. 47.6%, AOR = 7.77, p < .05), as compared to a control group who did not receive the internet content by email. If these preliminary findings can be confirmed by future research, free online content could be inexpensively distributed to at risk youth in underserved communities and could hold promise as an inexpensive method of HIV/STI prevention.

Magnetic Cytoskeleton Affinity Purification of Microtubule Motors Conjugated to Quantum Dots.

We develop magnetic cytoskeleton affinity (MiCA) purification, which allows for rapid isolation of molecular motors conjugated to large multivalent quantum dots, in miniscule quantities, which is especially useful for single-molecule applications. When purifying labeled molecular motors, an excess of fluorophores or labels is usually used. However, large labels tend to sediment during the centrifugation step of microtubule affinity purification, a traditionally powerful technique for motor purification. This is solved with MiCA, and purification time is cut from 2 h to 20 min, a significant time-savings when it needs to be done daily. For kinesin, MiCA works with as little as 0.6 µg protein, with yield of ∼27%, compared to 41% with traditional purification. We show the utility of MiCA purification in a force-gliding assay with kinesin, allowing, for the first time, simultaneous determination of whether the force from each motor in a multiple-motor system drives or hinders microtubule movement. Furthermore, we demonstrate rapid purification of just 30 ng dynein-dynactin-BICD2N-QD (DDB-QD), ordinarily a difficult protein-complex to purify.

Gender Differences in Emergency Department Visits and Detox Referrals for Illicit and Nonmedical Use of Opioids.

INTRODUCTION: Visits to the emergency department (ED) for use of illicit drugs and opioids have increased in the past decade. In the ED, little is known about how gender may play a role in drug-related visits and referrals to treatment. This study performs gender-based comparison analyses of drug-related ED visits nationwide. METHODS: We performed a cross-sectional analysis with data collected from 2004 to 2011 by the Drug Abuse Warning Network (DAWN). All data were coded to capture major drug categories and opioids. We used logistic regression models to find associations between gender and odds of referral to treatment programs. A second set of models were controlled for patient "seeking detox," or patient explicitly requesting for detox referral. RESULTS: Of the 27.9 million ED visits related to drug use in the DAWN database, visits by men were 2.69 times more likely to involve illicit drugs than visits by women (95% CI [2.56, 2.80]). Men were more likely than women to be referred to detox programs for any illicit drugs (OR 1.12, 95% CI [1.02-1.22]), for each of the major illicit drugs (e.g., cocaine: OR 1.27, 95% CI [1.15-1.40]), and for prescription opioids (OR 1.30, 95% CI [1.17-1.43]). This significant association prevailed after controlling for "seeking detox." CONCLUSION: Women are less likely to receive referrals to detox programs than men when presenting to the ED regardless of whether they are "seeking detox." Future research may help determine the cause for this gender-based difference and its significance for healthcare costs and health outcomes.

HIV Testing for At-Risk Adolescents at Rhode Island Hospital.

Early detection of HIV has great potential to reduce transmission, especially when newly diagnosed individuals are treated early. Early treatment and suppression of viral loads is known to effectively attenuate HIV transmission. However, little is known about whether persons at high risk for HIV are being appropriately tested during healthcare encounters according to national guidelines. Specifically, the at-risk adolescent population may be under tested and are not routinely monitored by state-level surveillance system. This study reviewed HIV testing rates for at-risk adolescents from 2005-2012 at the main tertiary care and pediatric center in Rhode Island. While the absolute number of HIV tests for at-risk adolescents continued to increase, the HIV testing rates for this population decreased during the seven year period. Increasing awareness of HIV testing for patients, their families, and physicians may improve the HIV testing rate among at-risk adolescents in Rhode Island.