Structural Insights into Pseudokinase Domains of Receptor Tyrosine Kinases.

Despite their apparent lack of catalytic activity, pseudokinases are essential signaling molecules. Here, we describe the structural and dynamic properties of pseudokinase domains from the Wnt-binding receptor tyrosine kinases (PTK7, ROR1, ROR2, and RYK), which play important roles in development. We determined structures of all pseudokinase domains in this family and found that they share a conserved inactive conformation in their activation loop that resembles the autoinhibited insulin receptor kinase (IRK). They also have inaccessible ATP-binding pockets, occluded by aromatic residues that mimic a cofactor-bound state. Structural comparisons revealed significant domain plasticity and alternative interactions that substitute for absent conserved motifs. The pseudokinases also showed dynamic properties that were strikingly similar to those of IRK. Despite the inaccessible ATP site, screening identified ATP-competitive type-II inhibitors for ROR1. Our results set the stage for an emerging therapeutic modality of "conformational disruptors" to inhibit or modulate non-catalytic functions of pseudokinases deregulated in disease.

ECM dimensionality tunes actin tension to modulate endoplasmic reticulum function and spheroid phenotypes of mammary epithelial cells.

Patient-derived organoids and cellular spheroids recapitulate tissue physiology with remarkable fidelity. We investigated how engagement with a reconstituted basement membrane in three dimensions (3D) supports the polarized, stress resilient tissue phenotype of mammary epithelial spheroids. Cells interacting with reconstituted basement membrane in 3D had reduced levels of total and actin-associated filamin and decreased cortical actin tension that increased plasma membrane protrusions to promote negative plasma membrane curvature and plasma membrane protein associations linked to protein secretion. By contrast, cells engaging a reconstituted basement membrane in 2D had high cortical actin tension that forced filamin unfolding and endoplasmic reticulum (ER) associations. Enhanced filamin-ER interactions increased levels of PKR-like ER kinase effectors and ER-plasma membrane contact sites that compromised calcium homeostasis and diminished cell viability. Consequently, cells with decreased cortical actin tension had reduced ER stress and survived better. Consistently, cortical actin tension in cellular spheroids regulated polarized basement membrane membrane deposition and sensitivity to exogenous stress. The findings implicate cortical actin tension-mediated filamin unfolding in ER function and underscore the importance of tissue mechanics in organoid homeostasis.

Novel Oridonin Analog CYD0682 Inhibits Hepatic Stellate Cell Activation via the Heat Shock Protein 90-Dependent STAT3 Pathway.

INTRODUCTION: Activated hepatic stellate cells (HSCs) are the primary effector cells in hepatic fibrosis, over depositing extracellular matrix (ECM) proteins. Our previous work found oridonin analog CYD0682 attenuates proliferation, Transforming Growth Factor ß (TGFß)-induced signaling, and ECM production in immortalized HSCs. The underlying mechanism behind these reductions is unclear. The Signal Transduction and Activator of Transcription 3 (STAT3) pathway plays a central role in HSC activation and has been found to be overexpressed in models of hepatic injury. In this study, we will examine the effect of CYD0682 on STAT3 signaling. METHODS: Immortalized human (LX-2) and rat (HSC-T6) HSC lines were treated with CYD0682 or Tanespimycin (17-AAG) with or without TGF-ß. Nuclear and cytosolic proteins were extracted. Protein expression was analyzed with Western blot. DNA binding activity was assessed with STAT3 DNA Binding ELISA. Cell viability was assessed with Alamar blue assay. RESULTS: CYD0682 treatment inhibited STAT3 phosphorylation at tyrosine 705 in a dose-dependent manner in LX-2 and HSC-T6 cells. STAT3 DNA binding activity and STAT3 regulated protein c-myc were significantly decreased by CYD0682. Notably, TGFß-induced STAT3 phosphorylation and ECM protein expression were inhibited by CYD0682. STAT3 is reported to be a Heat Shock Protein 90 (HSP90) client protein. Notably, CYD0682 attenuated the expression of endogenous STAT3 and other HSP90 client proteins FAK, IKKα, AKT and CDK9. HSP90 specific inhibitor 17-AAG suppressed endogenous and TGFß-induced STAT3 phosphorylation and ECM protein production. CONCLUSIONS: CYD0682 attenuates endogenous and TGFß-induced STAT3 activation and ECM production via an HSP90 dependent pathway in HSCs. Further study of this pathway may present new targets for therapeutic intervention in hepatic fibrosis.

Temporal Trends of Neonatal Surgical Conditions in Texas and Accessibility to Pediatric Surgical Care.

INTRODUCTION: Texas consistently accounts for approximately 10% of annual national births, the second highest of all US states. This temporal study aimed to evaluate incidences of neonatal surgical conditions across Texas and to delineate regional pediatric surgeon accessibility. METHODS: The Texas Birth Defects Registry was queried from 1999 to 2018, based on 11 well-established regions. Nine disorders (30,476 patients) were identified as being within the operative scope of pediatric surgeons: biliary atresia (BA), pyloric stenosis (PS), Hirschsprung's disease, stenosis/atresia of large intestine/rectum/anus, stenosis/atresia of small intestine, tracheoesophageal fistula/esophageal atresia, gastroschisis, omphalocele, and congenital diaphragmatic hernia. Annual and regional incidences were compared (/10,000 births). Statewide pediatric surgeons were identified through the American Pediatric Surgical Association directory. Regional incidences of neonatal disorder per surgeon were evaluated from 2010 to 2018 as a surrogate for provider disparity. RESULTS: PS demonstrated the highest incidence (14.405/10,000), while BA had the lowest (0.707/10,000). Overall, incidences of PS and BA decreased significantly, while incidences of Hirschsprung's disease and small intestine increased. Other diagnoses remained stable. Regions 2 (48.24/10,000) and 11 (47.79/10,000) had the highest incidence of neonatal conditions; Region 6 had the lowest (34.68/10,000). Three rural regions (#2, 4, 9) lacked pediatric surgeons from 2010 to 2018. Of regions with at least one surgeon, historically underserved regions (#10, 11) along the Texas-Mexico border consistently had the highest defect per surgeon rates. CONCLUSIONS: There are temporal and regional differences in incidences of neonatal conditions treated by pediatric surgeons across Texas. Improving access to neonatal care is a complex issue that necessitates collaborative efforts between state legislatures, health systems, and providers.

Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response.

Tumour cells evade immune surveillance by upregulating the surface expression of programmed death-ligand 1 (PD-L1), which interacts with programmed death-1 (PD-1) receptor on T cells to elicit the immune checkpoint response1,2. Anti-PD-1 antibodies have shown remarkable promise in treating tumours, including metastatic melanoma2-4. However, the patient response rate is low4,5. A better understanding of PD-L1-mediated immune evasion is needed to predict patient response and improve treatment efficacy. Here we report that metastatic melanomas release extracellular vesicles, mostly in the form of exosomes, that carry PD-L1 on their surface. Stimulation with interferon-γ (IFN-γ) increases the amount of PD-L1 on these vesicles, which suppresses the function of CD8 T cells and facilitates tumour growth. In patients with metastatic melanoma, the level of circulating exosomal PD-L1 positively correlates with that of IFN-γ, and varies during the course of anti-PD-1 therapy. The magnitudes of the increase in circulating exosomal PD-L1 during early stages of treatment, as an indicator of the adaptive response of the tumour cells to T cell reinvigoration, stratifies clinical responders from non-responders. Our study unveils a mechanism by which tumour cells systemically suppress the immune system, and provides a rationale for the application of exosomal PD-L1 as a predictor for anti-PD-1 therapy.

Activating mutations drive human MEK1 kinase using a gear-shifting mechanism.

There is an unmet need to classify cancer-promoting kinase mutations in a mechanistically cognizant way. The challenge is to understand how mutations stabilize different kinase configurations to alter function, and how this influences pathogenic potential of the kinase and its responses to therapeutic inhibitors. This goal is made more challenging by the complexity of the mutational landscape of diseases, and is further compounded by the conformational plasticity of each variant where multiple conformations coexist. We focus here on the human MEK1 kinase, a vital component of the RAS/MAPK pathway in which mutations cause cancers and developmental disorders called RASopathies. We sought to explore how these mutations alter the human MEK1 kinase at atomic resolution by utilizing enhanced sampling simulations and free energy calculations. We computationally mapped the different conformational stabilities of individual mutated systems by delineating the free energy landscapes, and showed how this relates directly to experimentally quantified developmental transformation potentials of the mutations. We conclude that mutations leverage variations in the hydrogen bonding network associated with the conformational plasticity to progressively stabilize the active-like conformational state of the kinase while destabilizing the inactive-like state. The mutations alter residue-level internal molecular correlations by differentially prioritizing different conformational states, delineating the various modes of MEK1 activation reminiscent of a gear-shifting mechanism. We define the molecular basis of conversion of this kinase from its inactive to its active state, connecting structure, dynamics, and function by delineating the energy landscape and conformational plasticity, thus augmenting our understanding of MEK1 regulation.

Computational studies of anaplastic lymphoma kinase mutations reveal common mechanisms of oncogenic activation.

Kinases play important roles in diverse cellular processes, including signaling, differentiation, proliferation, and metabolism. They are frequently mutated in cancer and are the targets of a large number of specific inhibitors. Surveys of cancer genome atlases reveal that kinase domains, which consist of 300 amino acids, can harbor numerous (150 to 200) single-point mutations across different patients in the same disease. This preponderance of mutations-some activating, some silent-in a known target protein make clinical decisions for enrolling patients in drug trials challenging since the relevance of the target and its drug sensitivity often depend on the mutational status in a given patient. We show through computational studies using molecular dynamics (MD) as well as enhanced sampling simulations that the experimentally determined activation status of a mutated kinase can be predicted effectively by identifying a hydrogen bonding fingerprint in the activation loop and the αC-helix regions, despite the fact that mutations in cancer patients occur throughout the kinase domain. In our study, we find that the predictive power of MD is superior to a purely data-driven machine learning model involving biochemical features that we implemented, even though MD utilized far fewer features (in fact, just one) in an unsupervised setting. Moreover, the MD results provide key insights into convergent mechanisms of activation, primarily involving differential stabilization of a hydrogen bond network that engages residues of the activation loop and αC-helix in the active-like conformation (in >70% of the mutations studied, regardless of the location of the mutation).

Predicting General Surgery Match Outcomes Using Standardized Ranking Metrics.

INTRODUCTION: Objective measurements for applicant ranking are becoming increasingly important, not only to help address the growing number of general surgery applicants each year but also to minimize bias and ensure consistency. We assessed if our general surgery applicant scoring system was an effective tool for accurately predicting the results of the resident match. METHODS: A retrospective review of applicant rank lists from 2017 to 2020 was conducted. Applicants were ranked based on the sum of preinterview and interview scores. The preinterview score is an objective metric related to the applicant's academic portfolio. The interview score is a standardized score based on interview performance. We reviewed match results from ranked candidates and categorized them as academic categorical (AC), community categorical (CC), preliminary surgical (PS), nonsurgical specialty (NS), or unmatched (UM) positions. RESULTS: A total of 378 applicants were interviewed. Forty-nine percent matched into AC, 22% into CC, 11% into PS, and 5% into NS positions, while 13% of the interviewees were UM. Applicants who matched into AC positions had significantly higher preinterview and interview scores than applicants in other categories. Applicants who matched into CC positions had significantly higher interview scores than those categorized as UM, but their preinterview scores did not differ significantly from the UM group. Applicants who did not match into a categorical position (PS, NS, or UM) did not have significantly different preinterview or interview scores from one another. CONCLUSIONS: Our standardized scoring system was effective in stratifying which applicants would match into categorical general surgery residency programs.

Prevalence and Impact of Infection during Extracorporeal Membrane Oxygenation in Oncologic Patients: A Retrospective Analysis of the Extracorporeal Life Support Organization (ELSO) Registry.

Background: Extracorporeal membrane oxygenation (ECMO) is widely utilized for severe cardiopulmonary insufficiency, but its application to the oncologic population has been debated given concern for increased risk of infection. This study aims to analyze the implications of infections acquired during ECMO runs in patients with malignancy. Methods: The Extracorporeal Life Support Organization (ELSO) database was queried for patients with an International Classification of Diseases code of neoplasms over the last two decades (2000-2019). Culture-proven infections during ECMO runs were analyzed and compared to previously reported data for all ECMO runs. Results: Two thousand, seven hundred and fifty-seven patients met inclusion criteria. Infection acquired during ECMO run was found in 687 patients, a significantly greater proportion compared to all ECMO runs (24.9% vs 11.7%; P = .001). Adult patients had a significantly higher rate of infection (27.0%; P < .001) compared to neonatal (11.0%) and pediatric (21.4%) patients. Prevalence of infection was highest in pulmonary ECMO (29.0%), while the infection rate standardized with ECMO duration was highest in extracorporeal cardiopulmonary resuscitation (55.03/1000-day ECMO run). Compared with ECMO for all diagnoses, the prevalence of Candida and Klebsiella infection was significantly higher in adult and pediatric oncologic patients. Regardless of the pathogen, the presence of infection was not associated with lower survival (38.6% vs 40.0%; P = .522). Conclusions: Oncologic patients had a significantly higher infection rate while on ECMO compared with the general ECMO population. However, the prognostic impact of these infections was minimal, thus ECMO should not be withheld in oncologic patients solely with concern for infection.

Crowding-induced membrane remodeling: Interplay of membrane tension, polymer density, architecture.

The plasma membrane hosts a wide range of biomolecules, mainly proteins and carbohydrates, that mediate cellular interactions with its environment. The crowding of such biomolecules regulates cellular morphologies and cellular trafficking. Recent discoveries have shown that the structure and density of cell surface polymers and hence the signaling machinery change with the state of the cell, especially in cancer progression. The alterations in membrane-attached glycocalyx and glycosylation of proteins and lipids are common features of cancer cells. The overexpression of glycocalyx polymers, such as mucin and hyaluronan, strongly correlates with cancer metastasis. Here, we present a mesoscale biophysics-based model that accounts for the shape regulation of membranes by crowding of membrane-attached biopolymer-glycocalyx and actin networks. Our computational model is based on the dynamically triangulated Monte Carlo model for membranes and coarse-grained representations of polymer chains. The model allows us to investigate the crowding-induced shape transformations in cell membranes in a tension- and graft polymer density-dependent manner. Our results show that the number of membrane protrusions and their shape depend on membrane tension, with higher membrane tension inducing more tubular protrusions than the vesicular shapes formed at low tension at high surface coverage of polymers. The shape transformations occur above the threshold density predicted by the polymer brush theory, but this threshold also depends on the membrane tension. Increasing the size of the polymer, either by changing the length or by adding side chains, is shown to increase the crowding-induced curvature. The effect of crowding is more prominent for flexible polymers than for semiflexible rigid polymers. We also present an extension of the model that incorporates properties of the actin-like filament networks and demonstrate how tubular structures can be generated by biopolymer crowding on the cytosolic side of cell membranes.

An interdomain helix in IRE1α mediates the conformational change required for the sensor's activation.

The unfolded protein response plays an evolutionarily conserved role in homeostasis, and its dysregulation often leads to human disease, including diabetes and cancer. IRE1α is a major transducer that conveys endoplasmic reticulum stress via biochemical signals, yet major gaps persist in our understanding of how the detection of stress is converted to one of several molecular outcomes. It is known that, upon sensing unfolded proteins via its endoplasmic reticulum luminal domain, IRE1α dimerizes and then oligomerizes (often visualized as clustering). Once assembled, the kinase domain trans-autophosphorylates a neighboring IRE1α, inducing a conformational change that activates the RNase effector domain. However, the full details of how the signal is transmitted are not known. Here, we describe a previously unrecognized role for helix αK, located between the kinase and RNase domains of IRE1α, in conveying this critical conformational change. Using constructs containing mutations within this interdomain helix, we show that distinct substitutions affect oligomerization, kinase activity, and the RNase activity of IRE1α differentially. Furthermore, using both biochemical and computational methods, we found that different residues at position 827 specify distinct conformations at distal sites of the protein, such as in the RNase domain. Of importance, an RNase-inactive mutant, L827P, can still dimerize with wildtype monomers, but this mutation inactivates the wildtype molecule and renders leukemic cells more susceptible to stress. We surmise that helix αK is a conduit for the activation of IRE1α in response to stress.

Quantification of Curvature Sensing Behavior of Curvature-Inducing Proteins on Model Wavy Substrates.

Curvature-inducing proteins are involved in a variety of membrane remodeling processes in the cell. Several in vitro experiments have quantified the curvature sensing behavior of these proteins in model lipid systems. One such system consists of a membrane bilayer laid atop a wavy substrate (Hsieh in Langmuir 28:12838-12843, 2012). In these experiments, the bilayer conforms to the wavy substrate, and curvature-inducing proteins show preferential segregation on the wavy membrane. Using a mesoscale computational membrane model based on the Helfrich Hamiltonian, here we present a study which analyzes the curvature sensing characteristics of this membrane-protein system, and elucidates key physical principles governing protein segregation on the wavy substrate and other in vitro systems. In this article we compute the local protein densities from the free energy landscape associated with membrane remodeling by curvature-inducing proteins. In specific, we use the Widom insertion technique to compute the free energy landscape for an inhomogeneous system with spatially varying density and the results obtained with this minimal model show excellent agreement with experimental studies that demonstrate the association between membrane curvature and local protein density. The free energy-based framework employed in this study can be used for different membrane morphologies and varied protein characteristics to gain mechanistic insights into protein sorting on membranes.

Reviewing ACGME Plastic Surgery Fellowship Case Logs: Is Surgical Experience Increasing?

INTRODUCTION: Over the last decade, there has been a 32% decrease in independent plastic surgery fellowships. The growing prevalence of 6-year integrated plastic surgery residencies, duty hour restrictions, and new subspecialty training fellowships for general surgeons have changed the training experience of plastic surgery fellows. METHODS: A retrospective review of the Accreditation Council for Graduate Medical Education (ACGME) case logs for graduating fellows of independent plastic surgery fellowships in the United States was conducted from 2011 to 2019. A linear regression analysis was conducted for each case log code and category, and a 95% level of confidence was assumed (α = 0.05). RESULTS: In 2011, 141 residents from 69 programs graduated with an average of 1469.7 cases. In 2019, 84 residents from 47 programs graduated with an average of 1952 cases. Index procedures significantly increased overall during the 9 y (P < 0.001). Categorical cases increased in esthetics (P < 0.001), including facelift, browlift, blepharoplasty, and more. Categorical cases increased in reconstructive surgery (P < 0.001), including treatment of deformities of the skin, lower extremities, and trunk, nerve decompression, and hand reconstruction. In breast procedures, an increase was seen in the reduction of mammoplasty, reconstruction, and treatment of other breast deformities. In head and neck procedures, an increase was seen in resection of head and neck neoplasms and secondary cleft lip repair. Decreases in procedural numbers were seen in primary cleft lip repair and hand reconstruction by primary closure. CONCLUSIONS: Despite a 32% decline in the number of independent plastic surgery fellowships over the last 9 y, plastic surgery fellows are obtaining significantly more surgical experience, both in esthetic and reconstructive surgery.

CVP and echo Measurements are Associated with Improved Outcomes in Patients with Gastrointestinal (GI) Hemorrhage: A Retrospective Analysis of the MIMIC- IV Database.

Purpose: Mortality associated with acute Gastrointestinal (GI) hemorrhage in intensive care units (ICU) has remained high in patients suffering from hemodynamic instability. Prompt recognition and rapid assessment of bleeding severity are crucial to improve survival. Central venous pressure (CVP) monitoring is commonly used for early recognition of intravascular imbalances, but its effectiveness in predicting fluid responsiveness is often questioned. Echocardiography (echo) is a rapid, noninvasive method to repeatedly assess cardiac function and fluid responsiveness. This study investigated the impact of CVP and echo measurements on the outcomes of critically ill patients with GI hemorrhage. Methods: The study was based on the Medical Information Mart for Intensive Care IV (MIMIC- IV) database. Patients were divided into four groups according to the usage of CVP and/or echo. The primary outcomes were 7-day, 14-day, 28-day, and overall mortalities after ICU admission. Cox Proportional-Hazards Models were used to elucidate the relationship between CVP/ Echo monitoring and mortality. The severity of illness of patients were adjusted by qSOFA score, SOFA score and base deficit level at admission. Results: Among 1705 eligible patients, 82 patients had both CVP and echo, 85 had CVP only, and 116 had Echo only. The results of survival analysis indicated that, comparing with those without either CVP or echo, the echo utilization was associated with improved mortalities at all time points during ICU stay for patients with moderate GI hemorrhage, and the combined use of CVP and echo was associated with lower 7-day,14-day and overall mortalities for patients with severe GI hemorrhage. Conclusion: Early usage of CVP and echo monitoring or echo alone are associated with lower mortality in the short and long-term when compared to patients without either measurement. Clinicians should consider goal-directed resuscitation guided by echo with/without CVP in patients with GI hemorrhage early after admission to ICU.

Outcome of Extracorporeal Membrane Oxygenation for Pediatric Patients With Neoplasm: An Extracorporeal Life Support Organization Database Study (2000-2019).

OBJECTIVES: This study update in usage and outcomes of pediatric extracorporeal membrane oxygenation (ECMO) for patients with neoplasm analyzed according to demographics, clinical variables, and complications. DESIGN: Retrospective database review of the Extracorporeal Life Support Organization registry from the last 2 decades (2000-2019). The data were divided between two decades in order to compare patients' backgrounds and outcomes over time. SETTING: ECMO centers reporting to Extracorporeal Life Support Organization. PATIENTS: Patients equal to or younger than 18 years old with International Classification of Diseases, 9th Revision and International Classification of Diseases, 10th Revision codes that referred to neoplasms who were managed with ECMO. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Demographics, cancer subtype, clinical variables, and ECMO complications were assessed in relation to the primary study outcome of survival to hospital discharge. Nine-hundred two patients met inclusion criteria; 699 patients were in the latest decade, which is more than three times the number from the previous decade (203 patients). On univariate analysis, compared with the previous decade, in the later decade, ECMO was more frequently applied in patients with pre-ECMO cardiac arrest (31.3% vs 17.1%; p < 0.001), and/or lower oxygenation index (38.0 vs 48.1; p < 0.001). We failed to identify a difference in survival between the 2 decades (42.8% vs 37.9%; p = 0.218). On multivariable analysis, diagnosis of hematologic malignancy, post-cardiopulmonary resuscitation support type, hematopoietic stem cell transplant, and age older than seven were each associated with greater odds of mortality. CONCLUSIONS: The use of ECMO in children with neoplasm has expanded over the latest decade with changes in patient selection. Mortality remains unchanged. Hence, although the clinician still should stay cautious in its application, ECMO can be considered as an option to rescue pediatric oncologic patients in the setting of worsening cardiopulmonary status in the PICU.

Surgical Jeopardy: Play to Learn.

BACKGROUND: General Surgery residencies use protected education time in various fashions in order to optimize content quality and yield for their learners. This knowledge is tested annually with the American Board for Surgery In-Training Examination (ABSITE) exam and is used to evaluate several aspects of a resident. We hypothesized that using a jeopardy game in educational conference would encourage residents to engage in self-learning and improve ABSITE scores at a single institution. MATERIALS AND METHODS: At a single institution, during protected education conference, residents played an hour-long surgical jeopardy game every 7 wk to summarize high yield topics discussed during the previous 6 wk of didactic learning. A 5-point Likert survey was completed by general surgery residents to discern the utility of the game format for learning. The ABSITE category scores were also evaluated from the year before and the year after the game was implemented. RESULTS: Twenty-four general surgery residents took the survey with >80% agreeing that the jeopardy format was either a fun or an effective way to learn general surgery topics. Additionally, over 80% of residents thought the game format helped with retention of knowledge. ABSITE categories that had a jeopardy session improved from 65.9% to 70.4% correct (P = 0.0003). ABSITE categories that did not have dedicated jeopardy had a non-significant increase in scores (67.7%-69.9%, P = 0.1). CONCLUSIONS: Implementing surgical jeopardy as a component of educational conferences in general surgery resident training is correlated with improvement of ABSITE scores. Surgical jeopardy may be easily adopted and implemented to stimulate self-directed learning for residents.

HJC0416 Attenuates Fibrogenesis in Activated Hepatic Stellate Cells via STAT3 and NF-κB Pathways.

BACKGROUND: Hepatic fibrosis is wound-healing response that is the result of hepatic stellate cell (HSC) activation and subsequent excess extracellular matrix deposition. HSCs can be activated by a variety of inflammatory stimuli as well as through the signal transducer and activator of transcription 3 (STAT3) pathway. HJC0416 is a novel, orally bioavailable small-molecule inhibitor of STAT3 that was developed by our team using a fragment-based drug design approach. Previously, our team has shown that HJC0416 has antifibrogenic effects in activated HSCs. Recently, increasing evidence suggests that nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) plays an important role in the activation of HSCs. In the present study, we examined the role of NF-κB inhibition of HSC activation by HJC0416. METHODS: LX-2 (human) and HSC-T6 (rat) cell lines were used. Expression levels of extracellular proteins, NF-κB and STAT3 expression and DNA binding, and inflammatory cytokine levels were determined using western blot, ELISA, and immunofluorescence assay. RESULTS: HJC0416 decreased cell viability in a dose-dependent manner in both cell lines and arrested the cell cycle at the S phase. Increased apoptosis was seen in LX-2 cells through Yo-Pro-1 and propidium iodide immunofluorescent stating. HJC0416 significantly decreased expression of fibronectin and collagen I as well as markedly decreased α-SMA and laminin. HJC0416 inhibited the STAT3 pathway by decreasing phosphorylation of STAT3, as well as signal transduction pathway activation. Notably, HJC0416 also inhibited the classic and alternative pathways of NF-κB activation. HJC0416 inhibited LPS-induced p65 nuclear translocation and DNA binding, as well as prevented phosphorylation and degradation of inhibitory protein IκBα. HJC0416 also prevented phosphorylation of serine residue 536 on p65. CONCLUSIONS: HJC0416, an inhibitor of STAT3, was found to have antifibrogenic properties in activated hepatic stellate cell lines. In addition, HJC0416 was found to inhibit the NF-κB pathway. Owing to this double effect, HJC0416 demonstrates promise for in vivo experimentation as an antifibrosis treatment.

Lateral distribution of phosphatidylinositol 4,5-bisphosphate in membranes regulates formin- and ARP2/3-mediated actin nucleation.

Spatial and temporal control of actin polymerization is fundamental for many cellular processes, including cell migration, division, vesicle trafficking, and response to agonists. Many actin-regulatory proteins interact with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and are either activated or inactivated by local PI(4,5)P2 concentrations that form transiently at the cytoplasmic face of cell membranes. The molecular mechanisms of these interactions and how the dozens of PI(4,5)P2-sensitive actin-binding proteins are selectively recruited to membrane PI(4,5)P2 pools remains undefined. Using a combination of biochemical, imaging, and cell biologic studies, combined with molecular dynamics and analytical theory, we test the hypothesis that the lateral distribution of PI(4,5)P2 within lipid membranes and native plasma membranes alters the capacity of PI(4,5)P2 to nucleate actin assembly in brain and neutrophil extracts and show that activities of formins and the Arp2/3 complex respond to PI(4,5)P2 lateral distribution. Simulations and analytical theory show that cholesterol promotes the cooperative interaction of formins with multiple PI(4,5)P2 headgroups in the membrane to initiate actin nucleation. Masking PI(4,5)P2 with neomycin or disrupting PI(4,5)P2 domains in the plasma membrane by removing cholesterol decreases the ability of these membranes to nucleate actin assembly in cytoplasmic extracts.

Dimerization and structure formation of colloids via capillarity at curved fluid interfaces.

Capillary interactions are ubiquitous between colloids trapped at fluid interfaces. Generally, colloids in fluid interfaces have pinned, undulated contact lines that distort the interface around them. To minimize the area, and therefore the energy of these distortions, colloids interact and assemble in a manner that depends on the shape of the host interface. On curved interfaces, capillary interactions direct isolated colloid motion along deviatoric curvature gradients. This directed motion relies on the leading order, long-ranged quadrupolar distortions made by the colloids' undulated pinned contact lines. Here we study pair interactions and dimer formation of colloids on non-uniformly curved fluid interfaces. Pair interaction energies are inferred to be order of 104kBT, and interacting forces are of order 10-1 pN for 10 micron particles adsorbed on interfaces formed around a 250 micron micropost. We compare experiments to analysis for the pair interaction energy, and identify criteria for dimers to form. We also study the formation of trapped structures by multiple particles to discern the influence of the underlying interface shape and the contact line undulations. By comparison to Monte Carlo simulations with potentials of interactions based on analysis, we find that higher order terms in the distortion fields generated by the particles play a major role in the structure formation on the curved interface. These interactions are determined by the particle's contact line and the host interface shape, and can be used to assemble particles independent of their material properties.

A multiscale biophysical model for the recruitment of actin nucleating proteins at the membrane interface.

The dynamics and organization of the actin cytoskeleton are crucial to many cellular events such as motility, polarization, cell shaping, and cell division. The intracellular and extracellular signaling associated with this cytoskeletal network is communicated through cell membranes. Hence the organization of membrane macromolecules and actin filament assembly are highly interdependent. Although the actin-membrane linkage is known to happen through many routes, the major class of interactions is through the direct interaction of actin-binding proteins with the lipid class containing poly-phosphatidylinositols (PPIs). Among the PPIs, phosphatidylinositol bisphosphate (PI(4,5)P2) acts as a significant factor controlling actin polymerization in the proximity of the membrane by binding to actin-associated proteins. The molecular interactions between these actin-binding proteins and the membrane lipids remain elusive. Here, using molecular modeling, analytical theory, and experimental methods, we investigate the binding of three different actin-binding proteins, mDia2, NWASP, and gelsolin, to membranes containing PI(4,5)P2 lipids. We perform molecular dynamics simulations on the protein-bilayer system and analyze the membrane binding in the form of hydrogen bonds and salt bridges at various PI(4,5)P2 and cholesterol concentrations. Our experimental study with PI(4,5)P2-containing large unilamellar vesicles mimics the computational experiments. Using the multivalencies of the proteins obtained in molecular simulations and the cooperative binding mechanisms of the proteins, we also propose a multivalent binding model that predicts the actin filament distributions at various PI(4,5)P2 and protein concentrations.