Synergy between Laminin-Derived Elastin-like Polypeptides (LELPs) Optimizes Cell Spreading.

A major component of the extracellular matrix (ECM), laminins, modulates cells via diverse receptors. Their fragments have emerging utility as components of "ECM-mimetics" optimized to promote cell-based therapies. Recently, we reported that a bioactive laminin peptide known as A99 enhanced cell binding and spreading via fusion to an elastin-like polypeptide (ELP).

The miRNA Landscape of Lacrimal Glands in a Murine Model of Autoimmune Dacryoadenitis.

Purpose: To analyze the changes in the lacrimal gland (LG) miRNAome from male nonobese diabetic (NOD) mice with autoimmune dacryoadenitis compared with LG from healthy male BALB/c and dacryoadenitis-free female NOD mice.

Methods: LG from these mice were collected for small RNA sequencing to identify dysregulated miRNAs; hits were validated by RT-qPCR in male NOD and BALB/c LG. Dysregulation of validated species within immune cell-enriched cell fractions and epithelial-enriched cell fractions from LG was probed by RT-qPCR.

Tear miRNAs Identified in a Murine Model of Sjögren's Syndrome as Potential Diagnostic Biomarkers and Indicators of Disease Mechanism.

Objective: The tear miRNAome of the male NOD mouse, a model of ocular symptoms of Sjögren's syndrome (SS), was analyzed to identify unique miRNAs.

Methods: Male NOD mice, aged 12-14 weeks, were used to identify tear miRNAs associated with development of autoimmune dacryoadenitis. Age- and sex-matched male BALB/c mice served as healthy controls while age-matched female NOD mice that do not develop the autoimmune dacryoadenitis characteristic of SS were used as additional controls.

Intracellular Dynamin Elastin-like Polypeptides Assemble into Rodlike, Spherical, and Reticular Dynasomes.

Dynamin (DNM) is a family of large GTPases possessing a unique mechanical ability to "pinch" off vesicles entering cells. DNM2 is the most ubiquitously expressed member of the DNM family. We developed a novel tool based on elastin-like polypeptide (ELP) technology to quickly, precisely, and reversibly modulate the structure of DNM2.

Small RNA Deep Sequencing Identifies a Unique miRNA Signature Released in Serum Exosomes in a Mouse Model of Sjögren's Syndrome.

Sjögren's Syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration and loss of function of moisture-producing exocrine glands as well as systemic inflammation. SS diagnosis is cumbersome, subjective and complicated by manifestation of symptoms that overlap with those of other rheumatic and ocular diseases. Definitive diagnosis averages 4-5 years and this delay may lead to irreversible tissue damage.

Caveolin elastin-like polypeptide fusions mediate temperature-dependent assembly of caveolar microdomains.

Caveolae are membrane organelles formed by submicron invaginations in the plasma membrane, and are involved in mechanosensing, cell signaling, and endocytosis. Although implicated broadly in physiology and pathophysiology, better tools are required to elucidate the precise role of caveolar processes through selective activation and inactivation of their trafficking. Our group recently reported that thermally-responsive elastin-like polypeptides (ELPs) can trigger formation of 'genetically engineered protein microdomains (GEPMs)' functionalized with either Clathrin-light chain or the epidermal growth factor receptor.

Evaluation of extracellular matrix mimetic laminin bioactive peptide and elastin-like polypeptide.

The extracellular matrix (ECM) is comprised of a large network of proteins that are essential for tissue development and repair. A bioactive RGD-containing peptide from laminin α1 chain, A99 (AGTFALRGDNPQG), promotes strong cell attachment and has demonstrated utility in cell culture and tissue engineering. Various materials can be utilized as a scaffold for bioactive peptides; however, it may be advantageous to design materials that use bioconjugation strategies that do not affect bioactivity, generate homogenous products, and can be produced at scale.

Nucleotide binding domain and leucine-rich repeat pyrin domain-containing protein 12: characterization of its binding to hematopoietic cell kinase.

Protein-protein interactions are key to define the function of nucleotide binding domain (NBD) and leucine-rich repeat (LRR) family, pyrin domain (PYD)-containing protein 12 (NLRP12). cDNA encoding the human PYD + NBD of NLRP12 was used as bait in a yeast two-hybrid screen with a human leukocyte cDNA library as prey. Hematopoiesis cell kinase (HCK), a member of the c-SRC family of non-receptor tyrosine kinases, was among the top hits.

Tears - more to them than meets the eye: why tears are a good source of biomarkers in Parkinson's disease.

Tears are a known source of biomarkers for both ocular and systemic diseases with particular advantages; specifically, the noninvasiveness of sample collection and a unique and increasingly better-defined protein composition. Here, we discuss our rationale for use of tears for discovery of biomarkers for Parkinson's disease (PD). These reasons include literature supporting changes in tear flow and composition in PD, and the interconnections between the ocular surface system and neurons affected in PD.

Levels of oligomeric α-Synuclein in reflex tears distinguish Parkinson's disease patients from healthy controls.

Due to active engagement of sensory and afferent nerve fibers in reflex tearing which could be affected in Parkinson's disease (PD), we tested reflex tears as a source of potential PD biomarkers. Reflex tears collected from 84 PD and 84 age- and sex-equivalent healthy controls (HC) were used to measure levels of oligomeric α-Syn (α-Syn), total α-Syn (α-Syn), CCL2, DJ-1, lactoferrin and MMP9. α-syn (p < 0.

Oligomeric α-synuclein is increased in basal tears of Parkinson's patients.

Secretion of proteins into basal tears of Parkinson's disease (PD) patients may be altered by changes in nerve function. Oligomeric α-Syn and total α-Syn, CCL-2, DJ-1, LF and MMP-9 were measured in basal tears from 93 PD patients and 82 age- and sex-equivalent healthy controls. α-Syn was decreased (p = 0.

A new temperature-dependent strategy to modulate the epidermal growth factor receptor.

The dynamic manipulation of kinases remains a major obstacle to unraveling cell-signaling networks responsible for the activation of biological systems. For example, epidermal growth factor (EGF) stimulates the epidermal growth factor receptor (EGFR/ErbB1); however, EGF also recruits other kinases (HER2/ErbB2) involved with various signaling pathways. To better study EGFR we report a new strategy to selectively activate receptor tyrosine kinases fused to elastin-like polypeptides (ELPs), which can be visualized inside mammalian cells using fixed and live-cell fluorescence microscopy.

Crosstalk of LKB1- and PTEN-regulated signals in liver morphogenesis and tumor development.

Unlabelled: Liver kinase B 1 (LKB1 or STK11) and PTEN (phosphatase and tensin homologue deleted on chromosome 10) are two tumor suppressors that regulate the mTOR signaling pathway. Deletion studies show that loss of either ( ) or ( ) leads to liver injury and development of hepatocarcinoma. In this study, we investigated the crosstalk of LKB1 and PTEN loss during tumorigenesis and liver development.

Regulation of Transporters and Channels by Membrane-Trafficking Complexes in Epithelial Cells.

The vectorial secretion and absorption of fluid and solutes by epithelial cells is dependent on the polarized expression of membrane solute transporters and channels at the apical and basolateral membranes. The establishment and maintenance of this polarized expression of transporters and channels are affected by divers protein-trafficking complexes. Moreover, regulation of the magnitude of transport is often under control of physiological stimuli, again through the interaction of transporters and channels with protein-trafficking complexes.

Single chain Fc-dimer-human growth hormone fusion protein for improved drug delivery.

Fc fusion protein technology has been successfully used to generate long-acting forms of several protein therapeutics. In this study, a novel Fc-based drug carrier, single chain Fc-dimer (sc(Fc)), was designed to contain two Fc domains recombinantly linked via a flexible linker. Since the Fc dimeric structure is maintained through the flexible linker, the hinge region was omitted to further stabilize it against proteolysis and reduce FcγR-related effector functions.

Imbalanced Rab3D versus Rab27 increases cathepsin S secretion from lacrimal acini in a mouse model of Sjögren's Syndrome.

The mechanism responsible for the altered spectrum of tear proteins secreted by lacrimal gland acinar cells (LGAC) in patients with Sjögren's Syndrome (SS) remains unknown. We have previously identified increased cathepsin S (CTSS) activity as a unique characteristic of tears of patients with SS. Here, we investigated the role of Rab3D, Rab27a, and Rab27b proteins in the enhanced release of CTSS from LGAC.

Flipping the Switch on Clathrin-Mediated Endocytosis using Thermally Responsive Protein Microdomains.

A ubiquitous approach to study protein function is to knock down activity (gene deletions, siRNA, small molecule inhibitors, etc) and study the cellular effects. Using a new methodology, this manuscript describes how to rapidly and specifically switch off cellular pathways using thermally responsive protein polymers. A small increase in temperature stimulates cytosolic elastin-like polypeptides (ELPs) to assemble microdomains.

Myosin IIB and F-actin control apical vacuolar morphology and histamine-induced trafficking of H-K-ATPase-containing tubulovesicles in gastric parietal cells.

Selective inhibitors of myosin or actin function and confocal microscopy were used to test the role of an actomyosin complex in controlling morphology, trafficking, and fusion of tubulovesicles (TV) containing H-K-ATPase with the apical secretory canaliculus (ASC) of primary-cultured rabbit gastric parietal cells. In resting cells, myosin IIB and IIC, ezrin, and F-actin were associated with ASC, whereas H-K-ATPase localized to intracellular TV. Histamine caused fusion of TV with ASC and subsequent expansion resulting from HCl and water secretion; F-actin and ezrin remained associated with ASC whereas myosin IIB and IIC appeared to dissociate from ASC and relocalize to the cytoplasm.

Evolution of preprofessional pharmacy curricula.

Objectives: To examine changes in preprofessional pharmacy curricular requirements and trends, and determine rationales for and implications of modifications.

Methods: Prerequisite curricular requirements compiled between 2006 and 2011 from all doctor of pharmacy (PharmD) programs approved by the Accreditation Council of Pharmacy Education were reviewed to ascertain trends over the past 5 years. An online survey was conducted of 20 programs that required either 3 years of prerequisite courses or a bachelor's degree, and a random sample of 20 programs that required 2 years of prerequisites.

Polymeric immunoglobulin receptor traffics through two distinct apically targeted pathways in primary lacrimal gland acinar cells.

The polymeric immunoglobulin receptor (pIgR) mediates transcytosis of dimeric immunoglobulin A (dIgA) and its release into mucosal secretions. The present study reveals the complexity of the trafficking of pIgR to the apical plasma membrane in epithelial cells with exocrine secretory functions; in rabbit lacrimal gland acinar cells (LGACs), trafficking of pIgR involves both the transcytotic pathway and one arm of the regulated secretory pathway. By specifically tracking pIgR endocytosed from the basolateral membrane, we show here that the Rab11a-regulated transcytotic pathway mediates the basal-to-apical transport of pIgR, and that pIgR sorted into the transcytotic pathway does not access the regulated secretory pathway.

A tunable and reversible platform for the intracellular formation of genetically engineered protein microdomains.

From mitochondria to the nuclear envelope, the controlled assembly of micro- and nanostructures is essential for life; however, the level at which we can deliberately engineer the assembly of microstructures within intracellular environments remains primitive. To overcome this obstacle, we present a platform to reversibly assemble genetically engineered protein microdomains (GEPMs) on the time scale of minutes within living cells. Biologically inspired from the human protein tropoelastin, these protein polymers form a secondary aqueous phase above a tunable transition temperature.

Adaptor protein complex 2-mediated, clathrin-dependent endocytosis, and related gene activities, are a prominent feature during maturation stage amelogenesis.

Molecular events defining enamel matrix removal during amelogenesis are poorly understood. Early reports have suggested that adaptor proteins (AP) participate in ameloblast-mediated endocytosis. Enamel formation involves the secretory and maturation stages, with an increase in resorptive function during the latter.