Technical Note: The Use of RNA-interference as a Tool to Find Proteins Involved in Melanosome Formation or Transport

Melanosomes are lysosome-related organelles that produce and transport the pigment melanin within melanocytes. Mutations in proteins required for melanosome transport and formation lead to a range of pigmentation defects, manifested at the cellular level as perinuclear clustering of melanosomes, or reduced sorting of melanosomal cargo such as tyrosinase-related protein 1 (TYRP1). A pilot screen was carried out to investigate whether a combination of cellular imaging and RNA interference could be used to identify new proteins involved in pigmentation pathways. In this study, eleven genes known to play a role in melanosome transport/formation or other pigmentation properties were knocked down in mouse melanocytes with shRNAmir constructs. The investigated genes were TYRP1, pallidin, cappuccino, dysbindin, HPS5, LYST, Myosin Va, melanophilin, RhoA, UBPY and mahogunin. In a blinded confocal imaging experiment, the only reproducible change observed in cells in which these targets were knocked down was a decrease in TYRP1 levels upon transfection with knockdown constructs against TYRP1 itself, or one of three constructs targeting HPS5 (Hermansky-Pudlak Syndrome 5). Upon analysis with high-content imaging software, only the knockdown construct against TYRP1 itself was detected. RT-PCR analysis showed that many of the shRNAmir constructs did not reduce mRNA and proteins levels enough to detect effects on melanosome properties. This was further examined for melanophilin, a protein necessary for melanosome transport. Altogether, the data show that this system is currently not sensitive enough for use in a screen for unknown regulators of melanosome transport and formation. The main obstacle appears to be incomplete reduction of target protein levels. Our observation that a ~50% reduction in mRNA level is not sufficient to elicit an effect is supported by the fact that heterozygous carriers of melanosomal transport disorders (Griscelli Syndrome, Hermansky-Pudlak Syndrome) do not display diseases phenotypes. A further reduction in protein levels, for example by viral infection of shRNA, may be required

Cyclic AMP increases COX-2 expression via mitogen-activated kinase in human myometrial cells

Cyclic AMP (cAMP) is the archetypal smooth muscle relaxant, mediating the effects of many hormones and drugs. However, recently PGI2, acting via cAMP/PKA, was found to increase contraction-associated protein expression in myometrial cells and to promote oxytocin-driven myometrial contractility. Cyclo-oxygenase-2 (COX-2) is the rate-limiting enzyme in prostaglandin synthesis, which is critical to the onset and progression of human labour. We have investigated the impact of cAMP on myometrial COX-2 expression, synthesis and activity. Three cAMP agonists (8-bromo-cAMP, forskolin and rolipram) increased COX-2 mRNA expression and further studies confirmed that this was associated with COX-2 protein synthesis and activity (increased PGE2 and PGI2 in culture supernatant) in primary cultures of human myometrial cells. These effects were neither reproduced by specific agonists nor inhibited by specific inhibitors of known cAMP-effectors (PKA, EPAC and AMPK). We then used shRNA to knockdown the same effectors and another recently described cAMP-effector PDZ-GEF1-2, without changing the response to cAMP. We found that MAPK activation mediated the cAMP effects on COX-2 expression and that PGE2 acts through EP-2 to activate MAPK and increase COX-2. These data provide further evidence in support of a dual role for cAMP in the regulation of myometrial function

Notch signaling in T cells is essential for allergic airway inflammation, but expression of the Notch ligands Jagged 1 and Jagged 2 on dendritic cells is dispensable

__Background:__ Allergic asthma is characterized by a TH2 response induced by dendritic cells (DCs) that present inhaled allergen. Although the mechanisms by which they instruct TH2 differentiation are still poorly understood, expression of the Notch ligand Jagged on DCs has been implicated in this process. __Objective:__ We sought to establish whether Notch signaling induced by DCs is critical for house dust mite (HDM)-driven allergic airway inflammation (AAI) in vivo. __Methods:__ The induction of Notch ligand expression on DC subsets by HDM was quantified by using quantitative real-time PCR. We used an HDM-driven asthma mouse model to compare the capacity of Jagged 1 and Jagged 2 single- and double-deficient DCs to induce AAI. In addition, we studied AAI in mice with a T cell-specific deletion of recombination signal-binding protein for immunoglobulin Jκ region (RBPJκ), a downstream effector of Notch signaling. __Results:__ HDM exposure promoted expression of Jagged 1, but not Jagged 2, on DCs. In agreement with published findings, in vitro-differentiated and HDM-pulsed Jagged 1 and Jagged 2 double-deficient DCs lacked the capacity to induce AAI. However, after in vivo intranasal sensitization and challenge with HDM, DC-specific Jagged 1 or Jagged 2 single- or double-deficient mice had eosinophilic airway inflammation and a TH2 cell activation phenotype that was not different from that in control littermates. In contrast, RBPJκ-def

Improving Navigation and Mapping with Arduino robot

During this Master Thesis work has been made on the Arduino robot to improve its general capabilities. Work has also been made to further explore the possibilities of the sensors used on all robots in the system. A hardware fault with the wheel armature cogs on the left side of the robot was discovered. This problem lead to the robot faultily estimating its position and be unable to drive in a straight line. This caused the robot to often drive into walls and not give reliable information about the mapping due to its corrupted position estimation. This problem was solved using a metal plate mounted on the armature to improve its stability and the tightness of each cog on the left side, so the cogs could not slip on each other. This solution completely removed the aforementioned problem and the robot now registers the correct wheel rotation. We do not expect a similar problem to occur on the right side as the work on the armature on that side is significantly better. Each sensor was searched for faults and tested for accuracy. The encoders were tested mainly by the wheel factor; the number converting encoder "ticks" to distance moved. This number was found to be adequate, however caution must be made as to the possibility of differing wheel factors depending on the surface. The surface tested on was room B333, "Slangelabben" at NTNU. The gyroscope was tested during rotation and compared to the encoder and the real-world value using the OptiTrack reflector-camera system. The gyroscope was found to be slightly off the real value each test. It was decided to poll for mean value more frequently to remove bias on the gyroscope on a more consistent basis. This seemed to improve the rotation estimation during our final tests. The compass was also tested during rotation and found to be very lacking. The noise factor on the sensor proved too great to adequately filter out during rotation. Due to this, the compass values had a negative impact on the heading estimation, and was removed from the system. It is advised that more work be done to explore the possibilities of filtering the compass and use it to reset gyroscope off-set. The accelerometer was found to give valuable, but very noisy data. Attempts were made at filtering and using the data, however none proved usable enough to directly implement on the current position estimation. It was decided to not be included in the software. More work is advised as the data is valuable and could potentially be used to detect slippage of the wheels and crashes. As the old navigation algorithm was not based on coordinates, but rather on distance and heading, it was decided that a new navigation algorithm be implemented in the robot. This algorithm is based on converting the heading and distance commands the server sends into specific coordinates, then use a similar controller to minimize the distance to the target. This controller is a two-step algorithm, which first rotates to the target, then drives forward while continuously controlling the heading towards the coordinates. A lot of focus was also put on smoother movements, as we witnessed a lot of overshoot on the robot both during rotation and forward movement due to the robot's mass. This new algorithm showed much greater results than expected and has almost eliminated the navigation error. During the final testing with the other robots it was shown to work well even if it showed some expected faults. The navigation is smoother and more reliable than before, and the position estimation is adequate based on its current algorithm

Notch signaling in T helper cell subsets: Instructor or unbiased amplifier?

For protection against pathogens, it is essential that naïve CD4+ T cells differentiate into specific effector T helper (Th) cell subsets following activation by antigen presented by dendritic cells (DCs). Next to T cell receptor and cytokine signals, membrane-bound Notch ligands have an important role in orchestrating Th cell differentiation. Several studies provided evidence that DC activation is accompanied by surface expression of Notch ligands. Intriguingly, DCs that express the delta-like or Jagged Notch ligands gain the capacity to instruct Th1 or Th2 cell polarization, respectively. However, in contrast to this model it has also been hypothesized that Notch signaling acts as a general amplifier of Th cell responses rather than an instructive director of specific T cell fates. In this alternative model, Notch enhances proliferation, cytokine production, and anti-apoptotic signals or promotes co-stimulatory signals in T cells. An instructive role for Notch ligand expressing DCs in the induction of Th cell differentiation is further challenged by evidence for the involvement of Notch signaling in differentiation of Th9, Th17, regulatory T cells, and follicular Th cells. In this review, we will discuss the two opposing models, referred to as the "instructive" and the "unbiased amplifier" model. We highlight both the function of different Notch receptors on CD4+ T cells and the impact of Notch ligands on antigen-presenting cells

High systemic IL-6 is associated with worse prognosis in patients with non-small cell lung cancer

Characteristic cytokine patterns have been described in different cancer patients and they are related to their diagnosis, prognosis, prediction of treatment responses and survival. A panel of cytokines was evaluated in the plasma of non-small cell lung cancer (NSCLC) patients and healthy controls to investigate their profile and relationship with clinical characteristics and overall survival. The case-controlled cross-sectional study design recruited 77 patients with confirmed diagnosis of NSCLC (cases) and 91 healthy subjects (controls) aimed to examine peripheral pro-inflammatory and anti-inflammatory cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, TNF and IFN-gamma) by Cytometry Beads Arrays (CBA Flex) in. The cytokine IL-6 showed a statistically significant difference among groups with increased expression in the case group (p < 0.001). The correlation between the cytokines expression with patient's clinical characteristics variables revealed the cytokine IL-6 was found to be associated with gender, showing higher levels in male (p = 0.036), whereas IL-17A levels were associated with TNM stage, being higher in III-IV stages (p = 0.044). We observed worse overall survival for individuals with high levels of IL-6 when compared to those with low levels of this cytokine in 6, 12 and 24 months. Further studies of IL-6 levels in independent cohort could clarify the real role of IL-6 as an independent marker of prognostic of NSCLC.Conselho Nacional de Desenvolvimento Científico e Tencnológico (CNPq) [Grant number 401775/2012-7 to ALF]; Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) [Grant number 2014/ 23414-8 to EMS]info:eu-repo/semantics/publishedVersio

Comparison of substrate specificity of the ubiquitin ligases Nedd4 and Nedd4-2 using proteome arrays

Target recognition by the ubiquitin system is mediated by E3 ubiquitin ligases. Nedd4 family members are E3 ligases comprised of a C2 domain, 2–4 WW domains that bind PY motifs (L/PPxY) and a ubiquitin ligase HECT domain. The nine Nedd4 family proteins in mammals include two close relatives: Nedd4 (Nedd4-1) and Nedd4L (Nedd4-2), but their global substrate recognition or differences in substrate specificity are unknown. We performed in vitro ubiquitylation and binding assays of human Nedd4-1 and Nedd4-2, and rat-Nedd4-1, using protein microarrays spotted with ∼8200 human proteins. Top hits (substrates) for the ubiquitylation and binding assays mostly contain PY motifs. Although several substrates were recognized by both Nedd4-1 and Nedd4-2, others were specific to only one, with several Tyr kinases preferred by Nedd4-1 and some ion channels by Nedd4-2; this was subsequently validated in vivo. Accordingly, Nedd4-1 knockdown or knockout in cells led to sustained signalling via some of its substrate Tyr kinases (e.g. FGFR), suggesting Nedd4-1 suppresses their signalling. These results demonstrate the feasibility of identifying substrates and deciphering substrate specificity of mammalian E3 ligases

Notch signaling is necessary for GATA3 function in the initiation of T cell development

GATA3 and Notch1 are essential for T cell development at the earliest stage, but their mutual roles in this process remain to be clarified. In this study, we demonstrated that impairment of T lymphopoiesis in hematopoietic progenitor cells (HPC) of GATA3-deficient fetal liver (FL) on day 11.5 of gestation (E11.5) was rescued only by introduction of both GATA3 and the intracellular region of Notch1 but not by either alone. However, the introduction of GATA3 only was sufficient for T cell induction in GATA3-deficient FL cells at the advanced stage, where Notch signaling is well detectable. This indicates that Notch signaling is necessary for GATA3 to function for T cell fate specification but is not sufficient without GATA3. On the other hand, Notch signaling is sufficient for blockage of B cell development without GATA3, suggesting that T cell fate specification at the branching point does not result simply from the developmental arrest of B cell lineage by Notch signaling.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/58545/1/977_ftp.pd