Imaging nuclear, endoplasmic reticulum, and plasma membrane events in real time

Live cell imaging can provide important information on cellular dynamics; however, the full utilisation of this technology has been hampered by the limitations of imaging reagents. Metal-based complexes have the potential to overcome many of the issues common to many current imaging agents. The rhenium (I)-based complex fac-[Re(CO)3(1,10-phenanthroline)(4-pyridyltetrazolate)], herein referred to as ReZolve-ER™, shows promise as a live cell imaging agent with rapid cell uptake, low cytotoxicity, resistance to photobleaching and compatibility with multicolour imaging. ReZolve-ER™ localised to the nuclear membrane/endoplasmic reticulum (ER) and allowed the detection of exocytotic events at the plasma membrane. Thus, we present a new imaging agent for monitoring live cell events in real time, which is ideal for imaging either short- or long-time courses

Investigating Intracellular Localisation and Cytotoxicity Trends for Neutral and Cationic Iridium Tetrazolato Complexes in Live Cells

A family of five neutral cyclometalated iridium(III) tetrazolato complexes and their methylated cationic analogues have been synthesised and characterised. The complexes are distinguished by variations of the substituents or degree of p conjugation on either the phenylpyridine or tetrazolato ligands. The photophysical properties of these species have been evaluated in organic and aqueous media, revealing predominantly a solvatochromic emission originating from mixed metal-to-ligand and ligand-to-ligand charge transfer excited states of triplet multiplicity. These emissions are characterised by typically long excited-state lifetimes (~hundreds of ns), and quantum yields around 5-10% in aqueous media. Methylation of the complexes caused a systematic red-shift of the emission profiles. The behaviour and the effects of the different complexes were then examined in cells. The neutral species localised mostly in the endoplasmic reticulum and lipid droplets, whereas the majority of the cationic complexes localised in the mitochondria. The amount of complexes found within cells does not depend on lipophilicity, which potentially suggests diverse uptake mechanisms. Methylated analogues were found to be more cytotoxic compared to the neutral species, a behaviour that might to be linked to a combination of uptake and intracellular localisation

CDKI-73 is a novel pharmacological inhibitor of Rab11 cargo delivery and innate immune secretion

Innate immunity is critical for host defence against pathogen and environmental challenge and this involves the production and secretion of immune mediators, such as antimicrobial peptides and pro-inflammatory cytokines. However, when dysregulated, innate immunity can contribute to multifactorial diseases, including inflammatory rheumatic disorders, type 2 diabetes, cancer, neurodegenerative and cardiovascular diseases and even septic shock. During an innate immune response, antimicrobial peptides and cytokines are trafficked via Rab11 multivesicular endosomes, and then sorted into Rab11 vesicles for traffic to the plasma membrane and secretion. In this study, a cyclin-dependent kinase inhibitor CDKI-73 was used to determine its effect on the innate immune response, based on previously identified targets for this compound. Our results showed that CDKI-73 inhibited the delivery of Rab11 vesicles to the plasma membrane, resulting in the accumulation of large multivesicular Rab11 endosomes near the cell periphery. In addition to the effect on endosome delivery, CDKI-73 down-regulated the amount of innate immune cargo, including the antimicrobial peptide Drosomycin and pro-inflammatory cytokines interleukin-6 (IL-6) and tumour necrosis factor alpha (TNFα). We concluded that CDKI-73 has the potential to regulate the delivery and secretion of certain innate immune cargo, which could be used to control inflammation.Alexandra Sorvina, Tetyana Shandala, Shudong Wang, David J. Sharkey, Emma Parkinson-Lawrence, Stavros Selemidis and Douglas A. Brook

A paradigm in immunochemistry, revealed by monoclonal antibodies to spatially distinct epitopes on Syntenin-1

Syntenin-1 is an essential multi-functional adaptor protein, which has multiple roles in membrane trafficking and exosome biogenesis, as well as scaffolding interactions with either the actin cytoskeleton or focal adhesions. However, how this functional multiplicity relates to syntenin-1 distribution in different endosome compartments or other intracellular locations and its underlying involvement in cancer pathogenesis have yet to be fully defined. To help facilitate the investigation of syntenin-1 biology, we developed two specific monoclonal antibodies (Synt-2C6 and Synt-3A11) to spatially distinct linear sequence epitopes on syntenin-1, which were each designed to be unique at the six-amino acid level. These antibodies produced very different intracellular staining patterns, with Synt-2C6 detecting endosomes and Synt-3A11 producing a fibrillar staining pattern suggesting a cytoskeletal localisation. Treatment of cells with Nocodazole altered the intracellular localisation of Synt-3A11, which was consistent with the syntenin-1 protein interacting with microtubules. In prostate tissue biopsies, Synt-3A11 defined atrophy and early-stage prostate cancer, whereas Synt-2C6 only showed minimal interaction with atrophic tissue. This highlights a critical need for site-specific antibodies and a knowledge of their reactivity to define differential protein distributions, interactions and functions, which may differ between normal and malignant cells.Ian R. D. Johnson, Alexandra Sorvina, Jessica M. Logan, Courtney R. Moore, Jessica K. Heatlie, Emma J. Parkinson-Lawrence, Stavros Selemidis, John J. O’Leary, Lisa M. Butler and Douglas A. Brook

Promotion of prophylaxis of HIV infection in Nizhnevartovsk: traditions and innovation

Article is devoted to the description of development of preventive activity in Nizhnevartovsk. The system of interaction between specialists of the AIDS Center, by the external organizations and groups of people pursuing a common goal - prophylaxis of HIV infection is in general presented. The main methodical approach uniting to all programs and the directions - the subject - subject'. This approach is innovative for a health system. It allows to develop creative activity, personal responsibility, an emotional and social maturity of the person according to age and situation in society, so it helps to solve problems of prophylaxis of HIV/AIDS more successfully.Статья посвящена описанию развития профилактической деятельности в городе Нижневартовске. Представлена система взаимодействия между специалистами Центра СПИД, внешними организациями и сообществами людей, преследующими общую цель - профилактику ВИЧ-инфекции. Основной методический подход, объединяющий все программы и направления - «субъект-субъектный». Данный подход является новаторским для системы здравоохранения Он позволяет развивать творческую активность, личную ответственность, эмоциональную и социальную зрелость личности в соответствии с возрастом и положением в обществе, что помогает более успешно решать задачи профилактики ВИЧ/СПИДа

Prediction of Prostate Cancer Biochemical and Clinical Recurrence Is Improved by IHC-Assisted Grading Using Appl1, Sortilin and Syndecan-1.

Gleason scoring is used within a five-tier risk stratification system to guide therapeutic decisions for patients with prostate cancer. This study aimed to compare the predictive performance of routine H&E or biomarker-assisted ISUP (International Society of Urological Pathology) grade grouping for assessing the risk of biochemical recurrence (BCR) and clinical recurrence (CR) in patients with prostate cancer. This retrospective study was an assessment of 114 men with prostate cancer who provided radical prostatectomy samples to the Australian Prostate Cancer Bioresource between 2006 and 2014. The prediction of CR was the primary outcome (median time to CR 79.8 months), and BCR was assessed as a secondary outcome (median time to BCR 41.7 months). The associations of (1) H&E ISUP grade groups and (2) modified ISUP grade groups informed by the Appl1, Sortilin and Syndecan-1 immunohistochemistry (IHC) labelling were modelled with BCR and CR using Cox proportional hazard approaches. IHC-assisted grading was more predictive than H&E for BCR (C-statistic 0.63 vs. 0.59) and CR (C-statistic 0.71 vs. 0.66). On adjusted analysis, IHC-assisted ISUP grading was independently associated with both outcome measures. IHC-assisted ISUP grading using the biomarker panel was an independent predictor of individual BCR and CR. Prospective studies are needed to further validate this biomarker technology and to define BCR and CR associations in real-world cohorts.Jessica M. Logan ... Lisa M. Butler ... Douglas A. Brooks ... et al

Options for financial support of local issues

The search for additional options for financing issues of local importance and the organization of economic activity of municipalities is particularly relevant in the context of the economic crisis and budgetary constraints in Russia. According to the author, the reason for the low activity of municipal-private partnership projects is often explained by the complexity of interaction between business and municipalities. It is obvious that only cooperation and interaction at all levels can ensure the solution of a number of problems, and, as a consequence, municipal development. The tool of such inter-municipal, inter-level, within the municipality cooperation with citizens and business can be a target bond loan for the implementation of a specific project. The author substantiates this hypothesis on the example of foreign practice, as well as considering a specific example of a possible municipality with the use of a bond and possible restrictions on the use of this tool

Drosophila Pkaap regulates Rab4/Rab11-dependent traffic and Rab11 exocytosis of innate immune cargo

The secretion of immune-mediators is a critical step in the host innate immune response to pathogen invasion, and Rab GTPases have an important role in the regulation of this process. Rab4/Rab11 recycling endosomes are involved in the sorting of immune-mediators into specialist Rab11 vesicles that can traffic this cargo to the plasma membrane; however, how this sequential delivery process is regulated has yet to be fully defined. Here, we report that Drosophila Pkaap, an orthologue of the human dual-specific A-kinase-anchoring protein 2 or D-AKAP2 (also called AKAP10), appeared to have a nucleotide-dependent localisation to Rab4 and Rab11 endosomes. RNAi silencing of pkaap altered Rab4/Rab11 recycling endosome morphology, suggesting that Pkaap functions in cargo sorting and delivery in the secretory pathway. The depletion of pkaap also had a direct effect on Rab11 vesicle exocytosis and the secretion of the antimicrobial peptide Drosomycin at the plasma membrane. We propose that Pkaap has a dual role in antimicrobial peptide traffic and exocytosis, making it an essential component for the secretion of inflammatory mediators and the defence of the host against pathogens