Investigating the role of proteins in melanosome transport

The specialized epidermal cells produce melanin within the organelles referred to as melanosomes. These melanosomes are believed to be transported along the microtubules from the perinuclear region to the actin and then the tri-partile complex of myosin Va, Melanophilin and Rab27a move the melanosomes along the actin filaments to the plasma membrane. Any defectiveness or disruption of these proteins leads to several disorders such as Hermansky-Pudlak Syndrome (HPS), Chediak-Higashi Syndrome and Griscelli Syndrome which are caused due to defects in genes that control organelle biogenesis, organelle motility or cargo transport within cells. The key aim of this project is to study the involvement of the proteins myosin Va, Rab27a and its effector proteins melanophilin and slp2-a. The experiment fluorescence Recovery after photobleaching(FRAP) was performed in different mutant cell lines such as melan-leaden (lacks melanophilin), melan-d (lacks myosin Va) and melan-ash (lacks Rab27a). We then transfected members of the tri-partile complex into these cell cells to observe their effect on melanosome transport. A mutant Rab27a which highly reduces the binding of its effectors slp2 and melanophilin was also used to investigate the binding dynamics of these effector proteins with Rab27a. Finally, these findings led to the idea of constructing a chimeric molecule where the RBD region of melanophilin been replaced with the RBD region of Slp2 and was expected to have a stable interaction with Rab27a. From these experiments, it was identified that melanophilin has a dynamic interaction with Rab27a and Slp2 has a stable interaction. It was understood that the Rab binding region(RBD) of slp2 and melanophilin perform a major part in controlling the nature of movement with Rab27a

The adaptor protein melanophilin regulates dynamic myosin-Va:cargo interaction and dendrite development in melanocytes

Regulation of organelle transport by the cytoskeleton is fundamental for eukaryotic survival. Cytoskeleton motors are typically modular proteins with conserved motor and diverse cargo binding domains. Motor:cargo interactions are often indirect and mediated by adaptor proteins e.g. Rab GTPases. Rab27a, via effector melanophilin (Mlph), recruits myosin-Va to melanosomes and thereby disperses them into melanocytes dendrites. To better understand how adaptors regulate motor:cargo interaction we used single melanosome fluorescence recovery after photo-bleaching (smFRAP) to characterise the association kinetics between myosin-Va, its adaptors and melanosomes. We found that myosin-Va and Mlph rapidly recovered after photo-bleaching, while Rab27a did not, indicating that myosin-Va and Mlph dynamically associate with melanosomes and Rab27a does not. This suggests that dynamic Rab27a:effector interaction rather than Rab27a melanosome:cytosol cycling regulates myosin-Va:melanosome association. Accordingly a Mlph-Rab27a fusion protein reduced myosin-Va smFRAP, indicating that it stabilised melanosomal myosin-Va. Finally, we tested the functional importance of dynamic myosin-Va:melanosome interaction. We found that while a myosin-Va-Rab27a fusion protein dispersed melanosomes in myosin-Va deficient cells, dendrites were significantly less elongated than in wild-type cells. Given that dendrites are the prime sites of melanosome transfer from melanocytes to keratinocytes we suggest that dynamic myosin-Va:melanosome interaction is important for pigmentation in vivo. Movie S1 Movie S1 MVa-tail expressed in wild-type (melan-a) cells (supports Figure 1). Movie S2 Movie S2 MVa-tail expressed in myosin-Va -/- (melan-d) cells (supports Figure S1A). Movie S3 Movie S3 MVa-FL expressed in myosin-Va -/- (melan-d) cells (supports Figure S1C). Movie S4 Movie S4 Rab27a expressed in wild-type (melan-a) cells (supports Figure 2A). Movie S5 Movie S5 Rab27a expressed in Rab27a -/- (melan-ash) cells (supports Figure 2B). Movie S6 Movie S6 Rab27a expressed in Mlph -/- (melan-ln) cells (supports Figure 2C). Movie S7 Movie S7 Rab27aSF1F4 expressed in wild-type (melan-a) cells (supports Figure 2D). Movie S8 Movie S8 Mlph expressed in wild-type (melan-a) cells (supports Figure 3A). Movie S9 Movie S9 Mlph expressed in Mlph -/- (melan-ln) cells (supports Figure 3B). Movie S10 Movie S10 Mlph expressed in myosin-Va -/- (melan-d) cells (supports Figure 3C). Movie S11 Movie S11 MVa-tail co-expressed with mCherry-Mlph expressed in Mlph -/- (melan-ln) cells (supports Figure 4B). Movie S12 Movie S12 MVa-tail co-expressed with mCherry-Mlph-Rab27aSF1F4 expressed in Mlph -/- (melan-ln) cells (supports Figure 4C). Movie S13 Movie S13 GFP-Mlph- Rab27aSF1F4 expressed in Mlph -/- (melan-ln) cells (supports Figure S4). Movie S14 Movie S14 Mlph R27BD expressed in wild-type (melan-a) cells (supports Figure S5). Movie S15 Movie S15 Myo-Rab expressed in myosin-Va -/- (melan-d) cells (supports Figures 5 and S7). Movie S16 Movie S16 Sytl2 (R27BD) expressed in wild-type (melan-a) cells (supports Figures S8).publishersversionpublishe

Proteomic profiling of human amnion for preterm birth biomarker discovery

Spontaneous preterm birth (PTB) complicates about 12% of pregnancies worldwide, remaining the main cause of neonatal morbidity and mortality. Spontaneous preterm birth PTBs is often caused by microbial-induced preterm labor, mediated by an inflammatory process threatening both maternal and newborn health. In search for novel predictive biomarkers of PTB and preterm prelabor rupture of the membranes (pPROM), and to improve understanding of infection related PTB, we performed an untargeted mass spectrometry discovery study on 51 bioptic mid zone amnion samples from premature babies. A total of 6352 proteins were identified. Bioinformatics analyses revealed a ranked core of 159 proteins maximizing the discrimination between the selected clinical stratification groups allowing to distinguish conditions of absent (FIR 0) from maximal Fetal Inflammatory Response (FIR 3) stratified in function of Maternal Inflammatory Response (MIR) grade. Matrix metallopeptidase-9 (MMP-9) was the top differentially expressed protein. Gene Ontology enrichment analysis of the core proteins showed significant changes in the biological pathways associated to inflammation and regulation of immune and infection response. Data suggest that the conditions determining PTB would be a transversal event, secondary to the maternal inflammatory response causing a breakdown in fetal-maternal tolerance, with fetal inflammation being more severe than maternal one. We also highlight matrix metallopeptidase-9 as a potential predictive biomarker of PTB that can be assayed in the maternal serum, for future investigation

Young professionals for health development: the Kenyan experience in combating non-communicable diseases

Young individuals (below 35 years) comprise an estimated 60% of the global population. Not only are these individuals currently experiencing chronic, non-communicable diseases (NCDs), either living with or at risk for these conditions, but will also experience the long-term repercussions of the current NCD policy implementations. It is thus imperative that they meaningfully contribute to the global discourse and responses for NCDs at the local level. Here, we profile one example of meaningful engagement: the Young Professionals Chronic Disease Network (YPCDN). The YPCDN is a global online network that provides a platform for young professionals to deliberate new and innovative methods of approaching the NCD challenges facing our societies. We provide a case study of the 2-year experiences of a country chapter (Kenya) of the YPCDN to demonstrate the significance and impact of emerging leaders in addressing the new global health agenda of the 21st century

Investigating the role of proteins in melanosome transport

The specialized epidermal cells produce melanin within the organelles referred to as melanosomes. These melanosomes are believed to be transported along the microtubules from the perinuclear region to the actin and then the tri-partile complex of myosin Va, Melanophilin and Rab27a move the melanosomes along the actin filaments to the plasma membrane. Any defectiveness or disruption of these proteins leads to several disorders such as Hermansky-Pudlak Syndrome (HPS), Chediak-Higashi Syndrome and Griscelli Syndrome which are caused due to defects in genes that control organelle biogenesis, organelle motility or cargo transport within cells. The key aim of this project is to study the involvement of the proteins myosin Va, Rab27a and its effector proteins melanophilin and slp2-a. The experiment fluorescence Recovery after photobleaching(FRAP) was performed in different mutant cell lines such as melan-leaden (lacks melanophilin), melan-d (lacks myosin Va) and melan-ash (lacks Rab27a). We then transfected members of the tri-partile complex into these cell cells to observe their effect on melanosome transport. A mutant Rab27a which highly reduces the binding of its effectors slp2 and melanophilin was also used to investigate the binding dynamics of these effector proteins with Rab27a. Finally, these findings led to the idea of constructing a chimeric molecule where the RBD region of melanophilin been replaced with the RBD region of Slp2 and was expected to have a stable interaction with Rab27a. From these experiments, it was identified that melanophilin has a dynamic interaction with Rab27a and Slp2 has a stable interaction. It was understood that the Rab binding region(RBD) of slp2 and melanophilin perform a major part in controlling the nature of movement with Rab27a

Bimetallic AC/Ag2CrO4/SnS heterostructure photoanode for energy conversion and storage: A self-powered Photocapacitor

The worldwide increase in generation of solar based electricity prompts the essentiality of research efforts on the development of energy storage systems. In this regard, self-powered photocapacitors are of keen interest as they can directly convert and store the solar energy in the form of electrical energy in a single device. This study reports the photoelectrochemical energy storage capacity of a novel photocapacitor fabricated with FTO/Activated Carbon (AC)/Ag2CrO4/SnS nanostructured photoanode. Initially, the Ag2CrO4 and SnS nanostructures were synthesized using simple ultrasonication technique and hydrothermal method, respectively. The crystallinity, morphology and optical properties of the synthesized nanostructures were then studied. The XRD patterns indicated orthorhombic structure of both Ag2CrO4 and SnS. Their optical band gaps were calculated as 1.93 and 1.65 eV, respectively using Kubelka-Munk plots. The FTO/AC/Ag2CrO4/SnS photoanode was then fabricated and photoelectrochemical studies, namely cyclic voltammetry and electrochemical impedance spectroscopy were carried out on a three electrode system. The FTO/AC/Ag2CrO4/SnS photoanode showed a specific capacitance of 4782 mF/g at the scan rate of 10 mVs−1 when the device was subjected to visible light illumination (1 sun). Hence, the fabricated heterostructured photoanode provides a promising path for the design and synthesis of novel highly efficient solar energy harvesting and storage materials as photocapacitors

Roles of Interfacial Modifiers in Inorganic Titania/Organic Poly(3-hexylthiophene) Heterojunction Hybrid Solar Cells

Hybrid Titanium dioxide/Poly(3-hexylthiophene) heterojunction solar cells have gained research interest as they have the potential to become cost-effective solar technology in the future. Limited power conversion efficiencies of about 5–6% have been reported so far, and an enhancement in efficiency was achieved through the engineering of the interface between Titanium dioxide (TiO2) and Poly(3-hexylthiophene) (P3HT). Evolution of this solar cell technology is relatively slow-moving due to the complex features of the metal oxide-polymer system and the limited understanding of the technology. In this review, we focus on recent developments in interface modified hybrid Titanium dioxide/Poly(3-hexylthiophene) solar cells, provide a short discussion on the working principle, device structure with interface modifiers, and summarize various types of interface modifiers studied to enhance the photovoltaic performance of hybrid TiO2/P3HT heterojunction solar cells. Further, we discuss the key factors influencing the power conversion efficiency and the role of a variety of interface modifiers in this regard. Finally, the challenges and perspectives related to hybrid TiO2/P3HT heterojunction solar cells are also explore