Almost 20 years of Cyclone; past, present and future

This paper discusses the history of the Cyclone library, whose purpose is to clone MAX/MSP objects into Pure Data and provide compatibility between these two environments. This paper describes the different phases and versions of Cyclone from its early days to the current state of development and our future plans

Almost 20 years of Cyclone; past, present and future

This paper discusses the history of the Cyclone library, whose purpose is to clone MAX/MSP objects into Pure Data and provide compatibility between these two environments. This paper describes the different phases and versions of Cyclone from its early days to the current state of development and our future plans

Knitting music and programming: Reflections on the frontiers of source code analysis

Source Code Analysis and Manipulation (SCAM) underpins virtually every operational software system. Despite the impact and ubiquity of SCAM principles and techniques in software engineering, there are still frontiers to be explored. Looking "inward" to existing techniques, one finds frontiers of performance, efficiency, accuracy, and usability, looking "outward" one finds new languages, new problems, and thus new approaches. This paper presents a reflective framework for characterizing source languages and domains. It draws on current research projects in music program analysis, musical score processing, and machine knitting to identify new frontiers for SCAM. The paper also identifies opportunities for SCAM to inspire, and be inspired by, problems and techniques in other domains

Sequestosome 1 is part of the interaction network of VAPB

VAPB (Vesicle-Associated-membrane Protein-associated protein B) is a tail-anchored membrane protein of the endoplasmic reticulum that can also be detected at the inner nuclear membrane. As a component of many contact sites between the endoplasmic reticulum and other organelles, VAPB is engaged in multiple protein interactions with a plethora of binding partners. A mutant version of VAPB, P56S-VAPB, which results from a single point mutation, is involved in a familial form of amyotrophic lateral sclerosis (ALS8). We performed RAPIDS (rapamycin- and APEX-dependent identification of proteins by SILAC) to identify proteins that interact with or are in close proximity to P56S-VAPB. The mutation abrogates the interaction of VAPB with many known binding partners. Here, we identify Sequestosome 1 (SQSTM1), a well-known autophagic adapter protein, as a major interaction/proximity partner of P56S-VAPB. Remarkably, not only the mutant protein, but also wild-type VAPB interacts with SQSTM1, as shown by proximity ligation assays and co-immunoprecipiation experiments

Decoding the Folding of Burkholderia glumae Lipase: Folding Intermediates En Route to Kinetic Stability

The lipase produced by Burkholderia glumae folds spontaneously into an inactive near-native state and requires a periplasmic chaperone to reach its final active and secretion-competent fold. The B. glumae lipase-specific foldase (Lif) is classified as a member of the steric-chaperone family of which the propeptides of α-lytic protease and subtilisin are the best known representatives. Steric chaperones play a key role in conferring kinetic stability to proteins. However, until present there was no solid experimental evidence that Lif-dependent lipases are kinetically trapped enzymes. By combining thermal denaturation studies with proteolytic resistance experiments and the description of distinct folding intermediates, we demonstrate that the native lipase has a kinetically stable conformation. We show that a newly discovered molten globule-like conformation has distinct properties that clearly differ from those of the near-native intermediate state. The folding fingerprint of Lif-dependent lipases is put in the context of the protease-prodomain system and the comparison reveals clear differences that render the lipase-Lif systems unique. Limited proteolysis unveils structural differences between the near-native intermediate and the native conformation and sets the stage to shed light onto the nature of the kinetic barrier

Untersuchung des Beitrags der Substratsammelantenne Sammelantenne zum Protonen/Laktat-Cotransport (in PfFNT und MCT1)

Some cells rely intensively on the coupled activity of glycolysis and lactate dehydrogenase to constantly generate ATP. This process releases lactate and protons, the accumulation of which would become detrimental to the cell´s survival. Therefore, the removal of these metabolites is critical to maintain the cells energy generation. This is ensured by monocarboxylate transporters (MCT), such as the human MCT and the Plasmodium falciparum formate nitrite transporter (PfFNT). In the case of human MCT, it has been established that their transport activity was modulated by partner proteins: its chaperone Basigin and carbonic anhydrases enzymes. The surface of such proteins act as proton and substrate collecting antennas, generating microenvironments of greater substrate concentration close to the transport sites. This work set out to investigate how such antennas were involved in the modulation of the monocarboxylate transport functionality of MCT1 and PfFNT. Initial attempts of expressing fusion constructs of carbonic anhydrase, Basigin chaperone and MCT1 transporter proved unsuccessful. Then, alternative methods of protein production were explored to observe interaction between the transporter and the proton antenna. Moreover, this work identified that C-terminal poly-Histidine tag initially intended for protein purification and identification would affect the transport capacity of such MCT1 transporter. This work also hypothesized that the PfFNT C-terminal helix, highly conserved among all human-infecting Plasmodia, plays the role of an endogenous proton collecting antenna facilitating the proton/lactate cotransport. Experimental results suggested that this terminus does modulate the substrate transport (radiolabeled lactate influx capacity was increased in acidic extracellular pH upon its deletion), but it remains to be determined whenever this collecting antenna increases the local concentration of protons or lactate

Retinal photoreceptor complement of paleognathous birds

Studies of the cone visual pigment complement from a wide range of representatives of the vertebrate classes have provided data on the origins and evolution of vertebrate colour vision. Tetrachromatic visual systems have been identified in many diurnal birds, as well as in some reptiles and fish. However, little data are available for the paleognathous birds, a group that represents the earliest offshoot of the main avian stem and may provide an important link between the visual system of reptiles and those of the neognathous birds. Microspectrophotometry (MSP) was used to determine the absorbance spectra of both rod and cone visual pigments and oil droplets from the retinae of the ostrich (Struthio camelus) and rhea (Rhea americana). Light and fluorescence microscopy of whole fresh tissue mounts was used to determine the relative numbers of oil droplets in the retinae. Both species possess rods, double cones and four classes of single cone identified by their oil droplets. The rods have [lambda]max at about 505-nm, whereas three cone pigments were recorded with [lambda]max at about 570, 505 and 445 nm. The 570-nm pigment is located in both members of the double cones and in a class of single cone containing a red (R-type) oil droplet with ?cut at about 560-nm. The 510-nm and 445-nm cone pigments were found in populations of single cones containing yellow (Y-type) and clear (C-type) oil droplets with ?cut at about 500 and 420-nm respectively. Double cones possess a pale (P-type) droplet in the principal member and a small droplet containing low concentrations of carotenoid (A-type) in the accessory member. The fourth class of single cone contains a transparent (T- type) droplet and in the ostrich a 405-nm pigment has been characterised. The complement of visual pigments and oil droplets, and the relative ratio of cone types in the ostrich and rhea are remarkably similar to that found in neognathous birds. Nucleotide sequences from opsin genes of both the ostrich and rhea have been identified. Comparison of these sequences with those from other avian species indicates that paleognathous opsins are highly homologous to the opsins found for neognathous avian species

Structural basis for the activation of the lipid scramblase TMEM16F

TMEM16F, a member of the conserved TMEM16 family, plays a central role in the initiation of blood coagulation and the fusion of trophoblasts. The protein mediates passive ion and lipid transport in response to an increase in intracellular Ca$^{2+}$. However, the mechanism of how the protein facilitates both processes has remained elusive. Here we investigate the basis for TMEM16F activation. In a screen of residues lining the proposed site of conduction, we identify mutants with strongly activating phenotype. Structures of these mutants determined herein by cryo-electron microscopy show major rearrangements leading to the exposure of hydrophilic patches to the membrane, whose distortion facilitates lipid diffusion. The concomitant opening of a pore promotes ion conduction in the same protein conformation. Our work has revealed a mechanism that is distinct for this branch of the family and that will aid the development of a specific pharmacology for a promising drug target

The interactions of viral matrix proteins with lipid membranes

This thesis describes the work undertaken to study the binding of lipid membranes by the viral matrix proteins hRSV-M and Influenza-A-M1. hRSV-M was recombinantly expressed and purified. It was the subjected to analysis by Langmuir-Blodgett trough experiments, Brewster angle microscopy, Confocal microscopy of giant unilamellar vesicles (GUVs), and binding studies with lipid nanodiscs. These studies showed hRSV-M having a preference for interacting with negatively charged lipids, namely phosphatidylserine, and for having different behaviours in Lo and Ld phases of membranes. During work on hRSV-M to improve its stability, it was discovered calcium stabilised the protein. This relationship was explored by ICPMS, differential scanning fluorimetry (DSF), circular dichroism (CD), mass spectrometry and microscale thermophoresis. This showed the hRSV-M is a calcium binding protein, containing two binding sites. Influenza-A-M1 was cloned into a plasmid vector and subsequently expressed and purified. The stability and structure of the protein was probed by DSF and CD measurements. The lipid interactions of this protein were then also explored by Langmuir-Blodgett trough isotherms and GUV binding under confocal microscopy. These showed that M1 is able to bind to phosphatidylserine containing membranes and causes vesicle budding from those membranes