Cell biological mechanisms of activity-dependent synapse to nucleus translocation of CRTC1 in neurons.

Previous studies have revealed a critical role for CREB-regulated transcriptional coactivator (CRTC1) in regulating neuronal gene expression during learning and memory. CRTC1 localizes to synapses but undergoes activity-dependent nuclear translocation to regulate the transcription of CREB target genes. Here we investigate the long-distance retrograde transport of CRTC1 in hippocampal neurons. We show that local elevations in calcium, triggered by activation of glutamate receptors and L-type voltage-gated calcium channels, initiate active, dynein-mediated retrograde transport of CRTC1 along microtubules. We identify a nuclear localization signal within CRTC1, and characterize three conserved serine residues whose dephosphorylation is required for nuclear import. Domain analysis reveals that the amino-terminal third of CRTC1 contains all of the signals required for regulated nucleocytoplasmic trafficking. We fuse this region to Dendra2 to generate a reporter construct and perform live-cell imaging coupled with local uncaging of glutamate and photoconversion to characterize the dynamics of stimulus-induced retrograde transport and nuclear accumulation

Gag Deadens but doesn't Mute the Crime: a Case Series of Homicidal Gagging

Background: Asphyxia is the commonest mode of death in various violent homicidal deaths and in majority of such cases, there may not be any evidence of external injury except the general features of asphyxia being the only proof to rely upon. Under such circumstances, even an experienced medicolegal expert may not go further than to declare the death to be due to asphyxia; the exact mode adopted being left unexplained.Case Report: We present here a case report of three family members who were killed by means of gagging with the motive of taking over the property.Conclusion: Deaths due to gagging is rare but most of the times homicidal. This case was peculiar as there were multiple individuals who were gagged simultaneously and their bodies were stuffed into trunk

Rapid degradation of mutant SLC25A46 by the ubiquitin-proteasome system results in MFN1/2-mediated hyperfusion of mitochondria.

SCL25A46 is a mitochondrial carrier protein that surprisingly localizes to the outer membrane and is distantly related to Ugo1. Here we show that a subset of SLC25A46 interacts with mitochondrial dynamics components and the MICOS complex. Decreased expression of SLC25A46 results in increased stability and oligomerization of MFN1 and MFN2 on mitochondria, promoting mitochondrial hyperfusion. A mutation at L341P causes rapid degradation of SLC25A46, which manifests as a rare disease, pontocerebellar hypoplasia. The E3 ubiquitin ligases MULAN and MARCH5 coordinate ubiquitylation of SLC25A46 L341P, leading to degradation by organized activities of P97 and the proteasome. Whereas outer mitochondrial membrane-associated degradation is typically associated with apoptosis or a specialized type of autophagy termed mitophagy, SLC25A46 degradation operates independently of activation of outer membrane stress pathways. Thus SLC25A46 is a new component in mitochondrial dynamics that serves as a regulator for MFN1/2 oligomerization. Moreover, SLC25A46 is selectively degraded from the outer membrane independently of mitophagy and apoptosis, providing a framework for mechanistic studies in the proteolysis of outer membrane proteins

Protein palmitoylation plays an important role in Trichomonas vaginalis adherence

The flagellated protozoan parasite Trichomonas vaginalis is the etiologic agent of trichomoniasis, the most common non-viral sexually transmitted infection worldwide. As an obligate extracellular pathogen, adherence to epithelial cells is critical for parasite survival within the human host and a better understanding of this process is a prerequisite for the development of therapies to combat infection. In this sense, recent work has shown S-acylation as a key modification that regulates pathogenesis in different protozoan parasites. However, there are no reports indicating whether this post-translational modification is a mechanism operating in T. vaginalis. In order to study the extent and function of S-acylation in T. vaginalis biology, we undertook a proteomic study to profile the full scope of S-acylated proteins in this parasite and reported the identification of 363 proteins involved in a variety of biological processes such as protein transport, pathogenesis related and signaling, among others. Importantly, treatment of parasites with the palmitoylation inhibitor 2-bromopalmitate causes a significant decrease in parasite: Parasite aggregation as well as adherence to host cells suggesting that palmitoylation could be modifying proteins that are key regulators of Trichomonas vaginalis pathogenesis.Fil: Nievas, Yésica Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Vashisht, Ajay A.. University of California; Estados UnidosFil: Corvi, Maria Martha. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Metz, Sebastián Darío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Johnson, Patricia J. University of California; Estados UnidosFil: Wohlschlegel, James A.. University of California; Estados UnidosFil: de Miguel, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentin

In Vivo Biotinylation of the Toxoplasma Parasitophorous Vacuole Reveals Novel Dense Granule Proteins Important for Parasite Growth and Pathogenesis.

UnlabelledToxoplasma gondii is an obligate intracellular parasite that invades host cells and replicates within a unique parasitophorous vacuole. To maintain this intracellular niche, the parasite secretes an array of dense granule proteins (GRAs) into the nascent parasitophorous vacuole. These GRAs are believed to play key roles in vacuolar remodeling, nutrient uptake, and immune evasion while the parasite is replicating within the host cell. Despite the central role of GRAs in the Toxoplasma life cycle, only a subset of these proteins have been identified, and many of their roles have not been fully elucidated. In this report, we utilize the promiscuous biotin ligase BirA* to biotinylate GRA proteins secreted into the vacuole and then identify those proteins by affinity purification and mass spectrometry. Using GRA-BirA* fusion proteins as bait, we have identified a large number of known and candidate GRAs and verified localization of 13 novel GRA proteins by endogenous gene tagging. We proceeded to functionally characterize three related GRAs from this group (GRA38, GRA39, and GRA40) by gene knockout. While Δgra38 and Δgra40 parasites showed no altered phenotype, disruption of GRA39 results in slow-growing parasites that contain striking lipid deposits in the parasitophorous vacuole, suggesting a role in lipid regulation that is important for parasite growth. In addition, parasites lacking GRA39 showed dramatically reduced virulence and a lower tissue cyst burden in vivo Together, the findings from this work reveal a partial vacuolar proteome of T. gondii and identify a novel GRA that plays a key role in parasite replication and pathogenesis.ImportanceMost intracellular pathogens reside inside a membrane-bound vacuole within their host cell that is extensively modified by the pathogen to optimize intracellular growth and avoid host defenses. In Toxoplasma, this vacuole is modified by a host of secretory GRA proteins, many of which remain unidentified. Here we demonstrate that in vivo biotinylation of proximal and interacting proteins using the promiscuous biotin ligase BirA* is a powerful approach to rapidly identify vacuolar GRA proteins. We further demonstrate that one factor identified by this approach, GRA39, plays an important role in the ability of the parasite to replicate within its host cell and cause disease

High Confidence Fission Yeast SUMO Conjugates Identified by Tandem Denaturing Affinity Purification

Covalent attachment of the small ubiquitin-like modifier (SUMO) to key targets in the proteome critically regulates the evolutionarily conserved processes of cell cycle control, transcription, DNA replication and maintenance of genome stability. The proteome-wide identification of SUMO conjugates in budding yeast has been invaluable in helping to define roles of SUMO in these processes. Like budding yeast, fission yeast is an important and popular model organism; however, the fission yeast Schizosaccharomyces pombe community currently lacks proteome-wide knowledge of SUMO pathway targets. To begin to address this deficiency, we adapted and used a highly stringent Tandem Denaturing Affinity Purification (TDAP) method, coupled with mass spectrometry, to identify fission yeast SUMO conjugates. Comparison of our data with that compiled in budding yeast reveals conservation of SUMO target enrichment in nuclear and chromatin-associated processes. Moreover, the SUMO "cloud" phenomenon, whereby multiple components of a single protein complex are SUMOylated, is also conserved. Overall, SUMO TDAP provides both a key resource of high confidence SUMO-modified target proteins in fission yeast, and a robust method for future analyses of SUMO function

Safety and efficacy of non-absorbable mesh in contemporary gynaecological surgery

Mesh-augmented pelvic floor surgery evolved to address the limitations of native tissue repair in reconstructive surgery. The development of the synthetic mid-urethral tape signalled a revolution in the treatment of stress urinary incontinence, whilst the use of mesh in abdominal apical prolapse repair may confer benefits over native tissue alternatives. However, these procedures can be associated with mesh-specific complications, underlining the need for shared decision-making between physicians and patients prior to mesh surgery. Transvaginal non-absorbable mesh implants for pelvic organ prolapse are associated with a high risk of serious adverse events, leading to withdrawal or restricted use in many countries. Increased scrutiny has led to growing concerns about complications associated with all types of mesh-augmented reconstructive surgery, attracting widespread media attention. National and international reports have been commissioned examining the safety and efficacy of mesh surgery in gynaecology. They have all highlighted systemic failures in the development, regulation and clinical adoption of medical devices. The widespread application of novel devices prior to the availability of reliable safety and efficacy data, and delayed recognition of adverse events, is of serious concern. Notwithstanding, the available data continue to support a role for mesh augmentation. This review outlines the evolution of gynaecological mesh, the safety and efficacy of pelvic floor surgery using non-absorbable mesh materials, and an overview of specific complications

Caenorhabditis elegans ALG-1 antimorphic mutations uncover functions for Argonaute in microRNA guide strand selection and passenger strand disposal

MicroRNAs are regulators of gene expression whose functions are critical for normal development and physiology. We have previously characterized mutations in a Caenorhabditis elegans microRNA-specific Argonaute ALG-1 (Argonaute-like gene) that are antimorphic [alg-1(anti)]. alg-1(anti) mutants have dramatically stronger microRNA-related phenotypes than animals with a complete loss of ALG-1. ALG-1(anti) miRISC (microRNA induced silencing complex) fails to undergo a functional transition from microRNA processing to target repression. To better understand this transition, we characterized the small RNA and protein populations associated with ALG-1(anti) complexes in vivo. We extensively characterized proteins associated with wild-type and mutant ALG-1 and found that the mutant ALG-1(anti) protein fails to interact with numerous miRISC cofactors, including proteins known to be necessary for target repression. In addition, alg-1(anti) mutants dramatically overaccumulated microRNA* (passenger) strands, and immunoprecipitated ALG-1(anti) complexes contained nonstoichiometric yields of mature microRNA and microRNA* strands, with some microRNA* strands present in the ALG-1(anti) Argonaute far in excess of the corresponding mature microRNAs. We show complex and microRNA-specific defects in microRNA strand selection and microRNA* strand disposal. For certain microRNAs (for example mir-58), microRNA guide strand selection by ALG-1(anti) appeared normal, but microRNA* strand release was inefficient. For other microRNAs (such as mir-2), both the microRNA and microRNA* strands were selected as guide by ALG-1(anti), indicating a defect in normal specificity of the strand choice. Our results suggest that wild-type ALG-1 complexes recognize structural features of particular microRNAs in the context of conducting the strand selection and microRNA* ejection steps of miRISC maturation