ResearchPad - signaling-molecules https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Mutations in <i>SPATA13/ASEF2</i> cause primary angle closure glaucoma]]> https://www.researchpad.co/article/elastic_article_15764 Glaucoma is the leading cause of irreversible blindness globally. Angle closure glaucoma accounts for 50% of all glaucoma blindness impacting quality of life and burden on health services. A number of variations in DNA appear to influence the risk of the disease. However, the biological mechanism underlying this important disease remains unclear. In this paper, we report the identification and functional characterisation of the first gene, mutation in which causes primary angle closure glaucoma in a seven generation Caucasian family. We have identified other variants in the same gene in another family and individuals with the disease. This gene is involved in cell division and is highly expressed in parts of the eye affected by the disease. Mutations in this gene appear to affect important enzyme activity involved in cell division. Identification of the disease-causing role of mutations in this gene helps to further the understanding of glaucoma aetiology and identifies potential therapeutic targets for disease management.

]]>
<![CDATA[C4-HSL aptamers for blocking qurom sensing and inhibiting biofilm formation in Pseudomonas aeruginosa and its structure prediction and analysis]]> https://www.researchpad.co/article/5c75abffd5eed0c484d07f98

This study aimed to screen DNA aptamers against the signal molecule C4-HSL of the rhl system for the inhibition of biofilm formation of Pseudomonas aeruginosa using an improved systematic evolution of ligand by exponential enrichment (SELEX) method based on a structure-switching fluorescent activating bead. The aptamers against the C4-HSL with a high affinity and specifity were successfully obtained and evaluated in real-time by this method. Results of biofilm inhibition experiments in vitro showed that the biofilm formation of P. aeruginosa was efficiently reduced to about 1/3 by the aptamers compared with that of the groups without the aptamers. Independent secondary structure simulation and computer-aided tertiary structure prediction (3dRNA) showed that the aptamers contained a highly conserved Y-shaped structural unit. Therefore, this study benefits the search for new methods for the detection and treatment of P. aeruginosa biofilm formation.

]]>
<![CDATA[Function and regulation of the Caenorhabditis elegans Rab32 family member GLO-1 in lysosome-related organelle biogenesis]]> https://www.researchpad.co/article/5bf3295ed5eed0c4843beab4

Cell type-specific modifications of conventional endosomal trafficking pathways lead to the formation of lysosome-related organelles (LROs). C. elegans gut granules are intestinally restricted LROs that coexist with conventional degradative lysosomes. The formation of gut granules requires the Rab32 family member GLO-1. We show that the loss of glo-1 leads to the mistrafficking of gut granule proteins but does not significantly alter conventional endolysosome biogenesis. GLO-3 directly binds to CCZ-1 and they both function to promote the gut granule association of GLO-1, strongly suggesting that together, GLO-3 and CCZ-1 activate GLO-1. We found that a point mutation in GLO-1 predicted to spontaneously activate, and function independently of it guanine nucleotide exchange factor (GEF), localizes to gut granules and partially restores gut granule protein localization in ccz-1(-) and glo-3(-) mutants. CCZ-1 forms a heterodimeric complex with SAND-1(MON1), which does not function in gut granule formation, to activate RAB-7 in trafficking pathways to conventional lysosomes. Therefore, our data suggest a model whereby the function of a Rab GEF can be altered by subunit exchange. glo-3(-) mutants, which retain low levels of GLO-3 activity, generate gut granules that lack GLO-1 and improperly accumulate RAB-7 in a SAND-1 dependent process. We show that GLO-1 and GLO-3 restrict the distribution of RAB-7 to conventional endolysosomes, providing insights into the segregation of pathways leading to conventional lysosomes and LROs.

]]>
<![CDATA[Release of GTP Exchange Factor Mediated Down-Regulation of Abscisic Acid Signal Transduction through ABA-Induced Rapid Degradation of RopGEFs]]> https://www.researchpad.co/article/5989da31ab0ee8fa60b84b27

The phytohormone abscisic acid (ABA) is critical to plant development and stress responses. Abiotic stress triggers an ABA signal transduction cascade, which is comprised of the core components PYL/RCAR ABA receptors, PP2C-type protein phosphatases, and protein kinases. Small GTPases of the ROP/RAC family act as negative regulators of ABA signal transduction. However, the mechanisms by which ABA controls the behavior of ROP/RACs have remained unclear. Here, we show that an Arabidopsis guanine nucleotide exchange factor protein RopGEF1 is rapidly sequestered to intracellular particles in response to ABA. GFP-RopGEF1 is sequestered via the endosome-prevacuolar compartment pathway and is degraded. RopGEF1 directly interacts with several clade A PP2C protein phosphatases, including ABI1. Interestingly, RopGEF1 undergoes constitutive degradation in pp2c quadruple abi1/abi2/hab1/pp2ca mutant plants, revealing that active PP2C protein phosphatases protect and stabilize RopGEF1 from ABA-mediated degradation. Interestingly, ABA-mediated degradation of RopGEF1 also plays an important role in ABA-mediated inhibition of lateral root growth. The presented findings point to a PP2C-RopGEF-ROP/RAC control loop model that is proposed to aid in shutting off ABA signal transduction, to counteract leaky ABA signal transduction caused by “monomeric” PYL/RCAR ABA receptors in the absence of stress, and facilitate signaling in response to ABA.

]]>
<![CDATA[Rac1 Activation Caused by Membrane Translocation of a Guanine Nucleotide Exchange Factor in Akt2-Mediated Insulin Signaling in Mouse Skeletal Muscle]]> https://www.researchpad.co/article/5989da1cab0ee8fa60b7d513

Insulin-stimulated glucose uptake in skeletal muscle is mediated by the glucose transporter GLUT4, which is translocated to the plasma membrane following insulin stimulation. Several lines of evidence suggested that the protein kinase Akt2 plays a key role in this insulin action. The small GTPase Rac1 has also been implicated as a regulator of insulin-stimulated GLUT4 translocation, acting downstream of Akt2. However, the mechanisms whereby Akt2 regulates Rac1 activity remain obscure. The guanine nucleotide exchange factor FLJ00068 has been identified as a direct regulator of Rac1 in Akt2-mediated signaling, but its characterization was performed mostly in cultured myoblasts. Here, we provide in vivo evidence that FLJ00068 indeed acts downstream of Akt2 as a Rac1 regulator by using mouse skeletal muscle. Small interfering RNA knockdown of FLJ00068 markedly diminished GLUT4 translocation to the sarcolemma following insulin administration or ectopic expression of a constitutively activated mutant of either phosphoinositide 3-kinase or Akt2. Additionally, insulin and these constitutively activated mutants caused the activation of Rac1 as shown by immunofluorescent microscopy using a polypeptide probe specific to activated Rac1 in isolated gastrocnemius muscle fibers and frozen sections of gastrocnemius muscle. This Rac1 activation was also abrogated by FLJ00068 knockdown. Furthermore, we observed translocation of FLJ00068 to the cell periphery following insulin stimulation in cultured myoblasts. Localization of FLJ00068 in the plasma membrane in insulin-stimulated, but not unstimulated, myoblasts and mouse gastrocnemius muscle was further affirmed by subcellular fractionation and subsequent immunoblotting. Collectively, these results strongly support a critical role of FLJ00068 in Akt2-mediated Rac1 activation in mouse skeletal muscle insulin signaling.

]]>
<![CDATA[Bistable front dynamics in a contractile medium: Travelling wave fronts and cortical advection define stable zones of RhoA signaling at epithelial adherens junctions]]> https://www.researchpad.co/article/5989db53ab0ee8fa60bdcdbf

Mechanical coherence of cell layers is essential for epithelia to function as tissue barriers and to control active tissue dynamics during morphogenesis. RhoA signaling at adherens junctions plays a key role in this process by coupling cadherin-based cell-cell adhesion together with actomyosin contractility. Here we propose and analyze a mathematical model representing core interactions involved in the spatial localization of junctional RhoA signaling. We demonstrate how the interplay between biochemical signaling through positive feedback, combined with diffusion on the cell membrane and mechanical forces generated in the cortex, can determine the spatial distribution of RhoA signaling at cell-cell junctions. This dynamical mechanism relies on the balance between a propagating bistable signal that is opposed by an advective flow generated by an actomyosin stress gradient. Experimental observations on the behavior of the system when contractility is inhibited are in qualitative agreement with the predictions of the model.

]]>
<![CDATA[IL-7 Receptor Mutations and Steroid Resistance in Pediatric T cell Acute Lymphoblastic Leukemia: A Genome Sequencing Study]]> https://www.researchpad.co/article/5989da43ab0ee8fa60b8abc9

Background

Pediatric acute lymphoblastic leukemia (ALL) is the most common childhood cancer and the leading cause of cancer-related mortality in children. T cell ALL (T-ALL) represents about 15% of pediatric ALL cases and is considered a high-risk disease. T-ALL is often associated with resistance to treatment, including steroids, which are currently the cornerstone for treating ALL; moreover, initial steroid response strongly predicts survival and cure. However, the cellular mechanisms underlying steroid resistance in T-ALL patients are poorly understood. In this study, we combined various genomic datasets in order to identify candidate genetic mechanisms underlying steroid resistance in children undergoing T-ALL treatment.

Methods and Findings

We performed whole genome sequencing on paired pre-treatment (diagnostic) and post-treatment (remission) samples from 13 patients, and targeted exome sequencing of pre-treatment samples from 69 additional T-ALL patients. We then integrated mutation data with copy number data for 151 mutated genes, and this integrated dataset was tested for associations of mutations with clinical outcomes and in vitro drug response. Our analysis revealed that mutations in JAK1 and KRAS, two genes encoding components of the interleukin 7 receptor (IL7R) signaling pathway, were associated with steroid resistance and poor outcome. We then sequenced JAK1, KRAS, and other genes in this pathway, including IL7R, JAK3, NF1, NRAS, and AKT, in these 69 T-ALL patients and a further 77 T-ALL patients. We identified mutations in 32% (47/146) of patients, the majority of whom had a specific T-ALL subtype (early thymic progenitor ALL or TLX). Based on the outcomes of these patients and their prednisolone responsiveness measured in vitro, we then confirmed that these mutations were associated with both steroid resistance and poor outcome.

To explore how these mutations in IL7R signaling pathway genes cause steroid resistance and subsequent poor outcome, we expressed wild-type and mutant IL7R signaling molecules in two steroid-sensitive T-ALL cell lines (SUPT1 and P12 Ichikawa cells) using inducible lentiviral expression constructs. We found that expressing mutant IL7R, JAK1, or NRAS, or wild-type NRAS or AKT, specifically induced steroid resistance without affecting sensitivity to vincristine or L-asparaginase. In contrast, wild-type IL7R, JAK1, and JAK3, as well as mutant JAK3 and mutant AKT, had no effect. We then performed a functional study to examine the mechanisms underlying steroid resistance and found that, rather than changing the steroid receptor’s ability to activate downstream targets, steroid resistance was associated with strong activation of MEK-ERK and AKT, downstream components of the IL7R signaling pathway, thereby inducing a robust antiapoptotic response by upregulating MCL1 and BCLXL expression. Both the MEK-ERK and AKT pathways also inactivate BIM, an essential molecule for steroid-induced cell death, and inhibit GSK3B, an important regulator of proapoptotic BIM. Importantly, treating our cell lines with IL7R signaling inhibitors restored steroid sensitivity. To address clinical relevance, we treated primary T-ALL cells obtained from 11 patients with steroids either alone or in combination with IL7R signaling inhibitors; we found that including a MEK, AKT, mTOR, or dual PI3K/mTOR inhibitor strongly increased steroid-induced cell death. Therefore, combining these inhibitors with steroid treatment may enhance steroid sensitivity in patients with ALL. The main limitation of our study was the modest cohort size, owing to the very low incidence of T-ALL.

Conclusions

Using an unbiased sequencing approach, we found that specific mutations in IL7R signaling molecules underlie steroid resistance in T-ALL. Future prospective clinical studies should test the ability of inhibitors of MEK, AKT, mTOR, or PI3K/mTOR to restore or enhance steroid sensitivity and improve clinical outcome.

]]>
<![CDATA[Activation of G Proteins by Guanine Nucleotide Exchange Factors Relies on GTPase Activity]]> https://www.researchpad.co/article/5989db24ab0ee8fa60bd0038

G proteins are an important family of signalling molecules controlled by guanine nucleotide exchange and GTPase activity in what is commonly called an ‘activation/inactivation cycle’. The molecular mechanism by which guanine nucleotide exchange factors (GEFs) catalyse the activation of monomeric G proteins is well-established, however the complete reversibility of this mechanism is often overlooked. Here, we use a theoretical approach to prove that GEFs are unable to positively control G protein systems at steady-state in the absence of GTPase activity. Instead, positive regulation of G proteins must be seen as a product of the competition between guanine nucleotide exchange and GTPase activity—emphasising a central role for GTPase activity beyond merely signal termination. We conclude that a more accurate description of the regulation of G proteins via these processes is as a ‘balance/imbalance’ mechanism. This result has implications for the understanding of intracellular signalling processes, and for experimental strategies that rely on modulating G protein systems.

]]>
<![CDATA[CDC-42 Orients Cell Migration during Epithelial Intercalation in the Caenorhabditis elegans Epidermis]]> https://www.researchpad.co/article/5989dabdab0ee8fa60baf703

Cell intercalation is a highly directed cell rearrangement that is essential for animal morphogenesis. As such, intercalation requires orchestration of cell polarity across the plane of the tissue. CDC-42 is a Rho family GTPase with key functions in cell polarity, yet its role during epithelial intercalation has not been established because its roles early in embryogenesis have historically made it difficult to study. To circumvent these early requirements, in this paper we use tissue-specific and conditional loss-of-function approaches to identify a role for CDC-42 during intercalation of the Caenorhabditis elegans dorsal embryonic epidermis. CDC-42 activity is enriched in the medial tips of intercalating cells, which extend as cells migrate past one another. Moreover, CDC-42 is involved in both the efficient formation and orientation of cell tips during cell rearrangement. Using conditional loss-of-function we also show that the PAR complex functions in tip formation and orientation. Additionally, we find that the sole C. elegans Eph receptor, VAB-1, functions during this process in an Ephrin-independent manner. Using epistasis analysis, we find that vab-1 lies in the same genetic pathway as cdc-42 and is responsible for polarizing CDC-42 activity to the medial tip. Together, these data establish a previously uncharacterized role for polarized CDC-42, in conjunction with PAR-6, PAR-3 and an Eph receptor, during epithelial intercalation.

]]>
<![CDATA[Rescaling of Spatio-Temporal Sensing in Eukaryotic Chemotaxis]]> https://www.researchpad.co/article/5989daf2ab0ee8fa60bc1caa

Eukaryotic cells respond to a chemoattractant gradient by forming intracellular gradients of signaling molecules that reflect the extracellular chemical gradient—an ability called directional sensing. Quantitative experiments have revealed two characteristic input-output relations of the system: First, in a static chemoattractant gradient, the shapes of the intracellular gradients of the signaling molecules are determined by the relative steepness, rather than the absolute concentration, of the chemoattractant gradient along the cell body. Second, upon a spatially homogeneous temporal increase in the input stimulus, the intracellular signaling molecules are transiently activated such that the response magnitudes are dependent on fold changes of the stimulus, not on absolute levels. However, the underlying mechanism that endows the system with these response properties remains elusive. Here, by adopting a widely used modeling framework of directional sensing, local excitation and global inhibition (LEGI), we propose a hypothesis that the two rescaling behaviors stem from a single design principle, namely, invariance of the governing equations to a scale transformation of the input level. Analyses of the LEGI-based model reveal that the invariance can be divided into two parts, each of which is responsible for the respective response properties. Our hypothesis leads to an experimentally testable prediction that a system with the invariance detects relative steepness even in dynamic gradient stimuli as well as in static gradients. Furthermore, we show that the relation between the response properties and the scale invariance is general in that it can be implemented by models with different network topologies.

]]>
<![CDATA[MafA Is Required for Postnatal Proliferation of Pancreatic β-Cells]]> https://www.researchpad.co/article/5989daa9ab0ee8fa60ba8ae1

The postnatal proliferation and maturation of insulin-secreting pancreatic β-cells are critical for glucose metabolism and disease development in adults. Elucidation of the molecular mechanisms underlying these events will be beneficial to direct the differentiation of stem cells into functional β-cells. Maturation of β-cells is accompanied by increased expression of MafA, an insulin gene transcription factor. Transcriptome analysis of MafA knockout islets revealed MafA is required for the expression of several molecules critical for β-cell function, including Glut2, ZnT8, Granuphilin, Vdr, Pcsk1 and Urocortin 3, as well as Prolactin receptor (Prlr) and its downstream target Cyclin D2 (Ccnd2). Inhibition of MafA expression in mouse islets or β-cell lines resulted in reduced expression of Prlr and Ccnd2, and MafA transactivated the Prlr promoter. Stimulation of β-cells by prolactin resulted in the phosphorylation and translocation of Stat5B and an increased nuclear pool of Ccnd2 via Prlr and Jak2. Consistent with these results, the loss of MafA resulted in impaired proliferation of β-cells at 4 weeks of age. These results suggest that MafA regulates the postnatal proliferation of β-cells via prolactin signaling.

]]>
<![CDATA[Cardiovascular Risks Associated with Low Dose Ionizing Particle Radiation]]> https://www.researchpad.co/article/5989da6fab0ee8fa60b942df

Previous epidemiologic data demonstrate that cardiovascular (CV) morbidity and mortality may occur decades after ionizing radiation exposure. With increased use of proton and carbon ion radiotherapy and concerns about space radiation exposures to astronauts on future long-duration exploration-type missions, the long-term effects and risks of low-dose charged particle irradiation on the CV system must be better appreciated. Here we report on the long-term effects of whole-body proton (1H; 0.5 Gy, 1 GeV) and iron ion (56Fe; 0.15 Gy, 1GeV/nucleon) irradiation with and without an acute myocardial ischemia (AMI) event in mice. We show that cardiac function of proton-irradiated mice initially improves at 1 month but declines by 10 months post-irradiation. In AMI-induced mice, prior proton irradiation improved cardiac function restoration and enhanced cardiac remodeling. This was associated with increased pro-survival gene expression in cardiac tissues. In contrast, cardiac function was significantly declined in 56Fe ion-irradiated mice at 1 and 3 months but recovered at 10 months. In addition, 56Fe ion-irradiation led to poorer cardiac function and more adverse remodeling in AMI-induced mice, and was associated with decreased angiogenesis and pro-survival factors in cardiac tissues at any time point examined up to 10 months. This is the first study reporting CV effects following low dose proton and iron ion irradiation during normal aging and post-AMI. Understanding the biological effects of charged particle radiation qualities on the CV system is necessary both for the mitigation of space exploration CV risks and for understanding of long-term CV effects following charged particle radiotherapy.

]]>
<![CDATA[Phospholipase Cε Modulates Rap1 Activity and the Endothelial Barrier]]> https://www.researchpad.co/article/5989db1eab0ee8fa60bcec68

The phosphoinositide-specific phospholipase C, PLCε, is a unique signaling protein with known roles in regulating cardiac myocyte growth, astrocyte inflammatory signaling, and tumor formation. PLCε is also expressed in endothelial cells, however its role in endothelial regulation is not fully established. We show that endothelial cells of multiple origins, including human pulmonary artery (HPAEC), human umbilical vein (HUVEC), and immortalized brain microvascular (hCMEC/D3) endothelial cells, express PLCε. Knockdown of PLCε in arterial endothelial monolayers decreased the effectiveness of the endothelial barrier. Concomitantly, RhoA activity and stress fiber formation were increased. PLCε-deficient arterial endothelial cells also exhibited decreased Rap1-GTP levels, which could be restored by activation of the Rap1 GEF, Epac, to rescue the increase in monolayer leak. Reintroduction of PLCε rescued monolayer leak with both the CDC25 GEF domain and the lipase domain of PLCε required to fully activate Rap1 and to rescue endothelial barrier function. Finally, we demonstrate that the barrier promoting effects PLCε are dependent on Rap1 signaling through the Rap1 effector, KRIT1, which we have previously shown is vital for maintaining endothelial barrier stability. Thus we have described a novel role for PLCε PIP2 hydrolytic and Rap GEF activities in arterial endothelial cells, where PLCε-dependent activation of Rap1/KRIT1 signaling promotes endothelial barrier stability.

]]>
<![CDATA[Gi Proteins Regulate Adenylyl Cyclase Activity Independent of Receptor Activation]]> https://www.researchpad.co/article/5989db04ab0ee8fa60bc7da9

Background and purpose

Despite the view that only β2- as opposed to β1-adrenoceptors (βARs) couple to Gi, some data indicate that the β1AR-evoked inotropic response is also influenced by the inhibition of Gi. Therefore, we wanted to determine if Gi exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes.

Experimental approach

We used the Gs-selective (R,R)- and the Gs- and Gi-activating (R,S)-fenoterol to selectively activate β2ARs (β1AR blockade present) in combination with Gi inactivation with pertussis toxin (PTX). We also determined the effect of PTX upon basal and forskolin-mediated responses. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats.

Key results

PTX amplified both the (R,R)- and (R,S)-fenoterol-evoked maximal inotropic response and concentration-dependent increases in cAMP accumulation. The EC50 values of fenoterol matched published binding affinities. The PTX enhancement of the Gs-selective (R,R)-fenoterol-mediated responses suggests that Gi regulates AC activity independent of receptor coupling to Gi protein. Consistent with this hypothesis, forskolin-evoked cAMP accumulation was increased and inotropic responses to forskolin were potentiated by PTX treatment. In non-PTX-treated tissue, phosphodiesterase (PDE) 3 and 4 inhibition or removal of either constitutive muscarinic receptor activation of Gi with atropine or removal of constitutive adenosine receptor activation with CGS 15943 had no effect upon contractility. However, in PTX-treated tissue, PDE3 and 4 inhibition alone increased basal levels of cAMP and accordingly evoked a large inotropic response.

Conclusions and implications

Together, these data indicate that Gi exerts intrinsic receptor-independent inhibitory activity upon AC. We propose that PTX treatment shifts the balance of intrinsic Gi and Gs activity upon AC towards Gs, enhancing the effect of all cAMP-mediated inotropic agents.

]]>
<![CDATA[Rapid Recycling of Ca2+ between IP3-Sensitive Stores and Lysosomes]]> https://www.researchpad.co/article/5989daddab0ee8fa60bba795

Inositol 1,4,5-trisphosphate (IP3) evokes release of Ca2+ from the endoplasmic reticulum (ER), but the resulting Ca2+ signals are shaped by interactions with additional intracellular organelles. Bafilomycin A1, which prevents lysosomal Ca2+ uptake by inhibiting H+ pumping into lysosomes, increased the amplitude of the initial Ca2+ signals evoked by carbachol in human embryonic kidney (HEK) cells. Carbachol alone and carbachol in combination with parathyroid hormone (PTH) evoke Ca2+ release from distinct IP3-sensitive Ca2+ stores in HEK cells stably expressing human type 1 PTH receptors. Bafilomycin A1 similarly exaggerated the Ca2+ signals evoked by carbachol or carbachol with PTH, indicating that Ca2+ released from distinct IP3-sensitive Ca2+ stores is sequestered by lysosomes. The Ca2+ signals resulting from store-operated Ca2+ entry, whether evoked by thapsigargin or carbachol, were unaffected by bafilomycin A1. Using Gd3+ (1 mM) to inhibit both Ca2+ entry and Ca2+ extrusion, HEK cells were repetitively stimulated with carbachol to assess the effectiveness of Ca2+ recycling to the ER after IP3-evoked Ca2+ release. Blocking lysosomal Ca2+ uptake with bafilomycin A1 increased the amplitude of each carbachol-evoked Ca2+ signal without affecting the rate of Ca2+ recycling to the ER. This suggests that Ca2+ accumulated by lysosomes is rapidly returned to the ER. We conclude that lysosomes rapidly, reversibly and selectively accumulate the Ca2+ released by IP3 receptors residing within distinct Ca2+ stores, but not the Ca2+ entering cells via receptor-regulated, store-operated Ca2+ entry pathways.

]]>
<![CDATA[A Low-Level Carbon Dioxide Laser Promotes Fibroblast Proliferation and Migration through Activation of Akt, ERK, and JNK]]> https://www.researchpad.co/article/5989daa2ab0ee8fa60ba6511

Background

Low-level laser therapy (LLLT) with various types of lasers promotes fibroblast proliferation and migration during the process of wound healing. Although LLLT with a carbon dioxide (CO2) laser was also reported to promote wound healing, the underlying mechanisms at the cellular level have not been previously described. Herein, we investigated the effect of LLLT with a CO2 laser on fibroblast proliferation and migration.

Materials and Methods

Cultured human dermal fibroblasts were prepared. MTS and cell migration assays were performed with fibroblasts after LLLT with a CO2 laser at various doses (0.1, 0.5, 1.0, 2.0, or 5.0 J/cm2) to observe the effects of LLLT with a CO2 laser on the proliferation and migration of fibroblasts. The non-irradiated group served as the control. Moreover, western blot analysis was performed using fibroblasts after LLLT with a CO2 laser to analyze changes in the activities of Akt, extracellular signal-regulated kinase (ERK), and Jun N-terminal kinase (JNK), which are signaling molecules associated with cell proliferation and migration. Finally, the MTS assay, a cell migration assay, and western blot analysis were performed using fibroblasts treated with inhibitors of Akt, ERK, or JNK before LLLT with a CO2 laser.

Results

In MTS and cell migration assays, fibroblast proliferation and migration were promoted after LLLT with a CO2 laser at 1.0 J/cm2. Western blot analysis revealed that Akt, ERK, and JNK activities were promoted in fibroblasts after LLLT with a CO2 laser at 1.0 J/cm2. Moreover, inhibition of Akt, ERK, or JNK significantly blocked fibroblast proliferation and migration.

Conclusions

These findings suggested that LLLT with a CO2 laser would accelerate wound healing by promoting the proliferation and migration of fibroblasts. Activation of Akt, ERK, and JNK was essential for CO2 laser-induced proliferation and migration of fibroblasts.

]]>
<![CDATA[Highly Sensitive Detection of Melamine Using a One-Step Sample Treatment Combined with a Portable Ag Nanostructure Array SERS Sensor]]> https://www.researchpad.co/article/5989db38ab0ee8fa60bd3f52

There is an urgent need for rapid and reliable methods able to detect melamine in animal feed. In this study, a quick, simple, and sensitive method for the determination of melamine content in animal feed was developed using surface-enhanced Raman spectroscopy on fabricated Ag nanorod (AgNR) array substrates with a one-step sample extraction procedure. The AgNR array substrates washed by HNO3 solvent (10−7 M) and methanol and showed the good stability within 6 months. The Raman shift at △ν = 682 cm−1 was used as the characteristic melamine peak in the calculations. Sufficient linearity was obtained in the 2–200 μg·g−1 range (R2 = 0.926). The limits of detection and quantification were 0.9 and 2 μg·g−1, respectively. The recovery rates were 89.7–93.3%, with coefficients of variation below 2.02%. The method showed good accuracy compared with the tradition GC-MS analysis. This new protocol only need 2 min to fininsh the detection which could be developed for rapid onsite screening of melamine contamination in quality control and market surveillance applications.

]]>
<![CDATA[Foramen Ovale Closure Is a Process of Endothelial-to-Mesenchymal Transition Leading to Fibrosis]]> https://www.researchpad.co/article/5989da40ab0ee8fa60b89dda

Patent foramen ovale (PFO) is an atrial septal deformity present in around 25% of the general population. PFO is associated with major causes of morbidity, including stroke and migraine. PFO appears to be heritable but genes involved in the closure of foramen ovale have not been identified. The aim of this study is to determine molecular pathways and genes that are responsible to the postnatal closure of the foramen ovale. Using Sprague-Dawley rat hearts as a model we analysed the dynamic histological changes and gene expressions at the foramen ovale region between embryonic day 20 and postnatal day 7. We observed a gradual loss of the endothelial marker PECAM1, an upregulation of the mesenchymal marker vimentin and α-smooth muscle actin, the elevation of the transcription factor Snail, and an increase of fibroblast activation protein (FAP) in the foramen ovale region as well as the deposition of collagen-rich connective tissues at the closed foramen ovale, suggesting endothelial-to-mesenchymal transition (EndMT) occurring during foramen ovale closure which leads to fibrosis. In addition, Notch1 and Notch3 receptors, Notch ligand Jagged1 and Notch effector HRT1 were highly expressed in the endocardium of the foramen ovale region during EndMT. Activation of Notch3 alone in an endothelial cell culture model was able to drive EndMT and transform endothelial cells to mesenchymal phenotype. Our data demonstrate for the first time that FO closure is a process of EndMT-mediated fibrosis, and Notch signalling is an important player participating in this process. Elucidation of the molecular mechanisms of the closure of foramen ovale informs the pathogenesis of PFO and may provide potential options for screening and prevention of PFO related conditions.

]]>
<![CDATA[Discovering Protein Receptors for Signaling Nucleotides]]> https://www.researchpad.co/article/5989d9ffab0ee8fa60b739c4 ]]> <![CDATA[Hybrid and Rogue Kinases Encoded in the Genomes of Model Eukaryotes]]> https://www.researchpad.co/article/5989d9fdab0ee8fa60b72a13

The highly modular nature of protein kinases generates diverse functional roles mediated by evolutionary events such as domain recombination, insertion and deletion of domains. Usually domain architecture of a kinase is related to the subfamily to which the kinase catalytic domain belongs. However outlier kinases with unusual domain architectures serve in the expansion of the functional space of the protein kinase family. For example, Src kinases are made-up of SH2 and SH3 domains in addition to the kinase catalytic domain. A kinase which lacks these two domains but retains sequence characteristics within the kinase catalytic domain is an outlier that is likely to have modes of regulation different from classical src kinases. This study defines two types of outlier kinases: hybrids and rogues depending on the nature of domain recombination. Hybrid kinases are those where the catalytic kinase domain belongs to a kinase subfamily but the domain architecture is typical of another kinase subfamily. Rogue kinases are those with kinase catalytic domain characteristic of a kinase subfamily but the domain architecture is typical of neither that subfamily nor any other kinase subfamily. This report provides a consolidated set of such hybrid and rogue kinases gleaned from six eukaryotic genomes–S.cerevisiae, D. melanogaster, C.elegans, M.musculus, T.rubripes and H.sapiens–and discusses their functions. The presence of such kinases necessitates a revisiting of the classification scheme of the protein kinase family using full length sequences apart from classical classification using solely the sequences of kinase catalytic domains. The study of these kinases provides a good insight in engineering signalling pathways for a desired output. Lastly, identification of hybrids and rogues in pathogenic protozoa such as P.falciparum sheds light on possible strategies in host-pathogen interactions.

]]>