Plasmodial Hsp40s: New avenues for antimalarial drug discovery

Malaria, an infectious disease caused by Plasmodium spp, is one of the world\u27s most dangerous diseases, accounting for more than half a million deaths yearly. The long years of co-habitation between the parasite and its hosts (human and mosquito), is a testimony to the parasite’s ability to escape the immune system and develop drug resistance mechanisms. Currently, an important search area for improved pharmacotherapy are molecular chaperones of the heat shock protein family, abundant in Plasmodium falciparum and contributing to its continuous survival and development. Thus far, small molecule inhibitor studies on P. falciparum Hsp70s and Hsp90s have indicated that they are promising antimalarial targets. However, not much attention has been given to Hsp40s as potential antimalarial drug targets. Hsp40s are known to function as chaperones by preventing protein aggregation, and as co-chaperones, by regulating the chaperone activities of Hsp70s to ensure proper protein folding. There are only a limited number of reviews on Hsp40s as drug targets, and the few reviews on plasmodial Hsp40s tend to focus largely on the intra-erythrocytic stage of the parasite life cycle. Therefore, this review will summarize what is known about Hsp40s throughout the malaria parasite life cycle, and critically evaluate their potential to serve as new avenues for antimalarial drug discovery

Assessment of potential anti-cancer stem cell activity of marine algal compounds using an in vitro mammosphere assay

Background: The cancer stem cell (CSC) theory proposes that tumours arise from and are sustained by a subpopulation of cells with both cancer and stem cell properties. One of the key hallmarks of CSCs is the ability to grow anchorage-independently under serum-free culture conditions resulting in the formation of tumourspheres. It has further been reported that these cells are resistant to traditional chemotherapeutic agents. Methods: In this study, the tumoursphere assay was validated in MCF-7 cells and used to screen novel marine algal compounds for potential anti-cancer stem cell (CSC) activity in vitro. Results: MCF-7 breast cancer cells were observed to generate tumourspheres or mammospheres after 3-5 days growth in anchorage-independent conditions and an apparent enrichment in potential CSCs was observed by an increase in the proportion of CD44high/CD24low marker-bearing cells and Oct4 expression compared to those in the bulk population grown in regular adherent conditions. Using this assay, a set of algal metabolites was screened for the ability to inhibit mammosphere development as a measure of potential anti-CSC activity. We report that the polyhalogenated monoterpene stereoisomers RU017 and RU018 isolated from the red alga Plocamium cornutum, both of which displayed no cytotoxicity against either adherent MCF-7 breast cancer or MCF-12A non-transformed breast epithelial cells, were able to prevent MCF-7 mammosphere formation in vitro. On the other hand, neither the brown algal carotenoid fucoxanthin nor the chemotherapeutic paclitaxel, both of which were toxic to adherent MCF-7 and MCF-12A cells, were able to inhibit mammosphere formation. In fact, pre-treatment with paclitaxel appeared to enhance mammosphere formation and development, a finding which is consistent with the reported resistance of CSCs to traditional chemotherapeutic agents. Conclusion: Due to the proposed clinical significance of CSC in terms of tumour initiation and metastasis, the identification of agents able to inhibit this subpopulation has clinical significance

Assessment of potential anti-cancer stem cell activity of marine algal compounds using an in vitro mammosphere assay:

The cancer stem cell (CSC) theory proposes that tumours arise from and are sustained by a subpopulation of cells with both cancer and stem cell properties. One of the key hallmarks of CSCs is the ability to grow anchorage-independently under serum-free culture conditions resulting in the formation of tumourspheres. It has further been reported that these cells are resistant to traditional chemotherapeutic agents

Evaluation of pre-hospital use of a valsalva assist device in the emergency treatment of supraventricular tachycardia [EVADE]: a randomised controlled feasibility trial

Background: The valsalva manoeuvre is an internationally recommended initial treatment for supraventricular tachycardia (SVT). The pre-hospital use of a valsalva assist device, to help deliver this manoeuvre, could improve cardioversion rates and reduce the need for patients to attend hospital. Methods: We conducted a randomised controlled feasibility trial comparing ambulance clinician use of a valsalva assist device versus standard care to treat adult patients presenting to an ambulance service in the south west of England. Eligible consenting participants were randomised 1:1 to device or standard care with trial procedures mirroring a proposed definitive trial.Feasibility was assessed upon ambulance clinician and participant recruitment rates and feedback, data completeness and potential future primary outcome rates. Results: Over a 6 months period (1 July to 31 December 2018), 276 (23%) of 1183 eligible ambulance clinicians were trained and they recruited 34 participants; approximately 10% of patients presenting with suspected SVT during that time. Seventeen participants were randomised to each arm and all underwent their allocated valsalva strain method. All trial data and 63/68 (93%) of pre and post-valsalva ECGs were available. Seven (21%) participants had ineligible initial rhythms on retrospective expert ECG review. Valsalva assist device use was associated with cardioversion and non-conveyance in 4 (24%) and 2 (12%) participants respectively. No participants assigned to standard care were cardioverted and all were conveyed. Participant feedback highlighted the challenges of retaining trial information during an SVT attack. Conclusions: The trial achieved efficient clinician training, randomisation and data collection, and there was an encouraging effect signal associated with device use. However, trial design changes should be considered to address the relatively small proportion of eligible patients recruited and challenges identified with consent and confirmation of cardioversion as a primary outcome. Trial registration: The trial was registered with ClinicalTrials.gov (NCT03514628) on 2 May 2018.This article is freely available via Open Access. Click on the Publisher URL to access it via the publisher's site.The study was funded by a grant from SWASFT (Ref: 16-019) from research capacity funding provided by the National Institute for Health Research (NIHR)published version, accepted versio

Emergence and Modular Evolution of a Novel Motility Machinery in Bacteria

Bacteria glide across solid surfaces by mechanisms that have remained largely mysterious despite decades of research. In the deltaproteobacterium Myxococcus xanthus, this locomotion allows the formation stress-resistant fruiting bodies where sporulation takes place. However, despite the large number of genes identified as important for gliding, no specific machinery has been identified so far, hampering in-depth investigations. Based on the premise that components of the gliding machinery must have co-evolved and encode both envelope-spanning proteins and a molecular motor, we re-annotated known gliding motility genes and examined their taxonomic distribution, genomic localization, and phylogeny. We successfully delineated three functionally related genetic clusters, which we proved experimentally carry genes encoding the basal gliding machinery in M. xanthus, using genetic and localization techniques. For the first time, this study identifies structural gliding motility genes in the Myxobacteria and opens new perspectives to study the motility mechanism. Furthermore, phylogenomics provide insight into how this machinery emerged from an ancestral conserved core of genes of unknown function that evolved to gliding by the recruitment of functional modules in Myxococcales. Surprisingly, this motility machinery appears to be highly related to a sporulation system, underscoring unsuspected common mechanisms in these apparently distinct morphogenic phenomena

The malarial exported PFA0660w is an Hsp40 co-chaperone of PfHsp70-x

Plasmodium falciparum, the human pathogen responsible for the most dangerous malaria infection, survives and develops in mature erythrocytes through the export of proteins needed for remodelling of the host cell. Molecular chaperones of the heat shock protein (Hsp) family are prominent members of the exportome, including a number of Hsp40s and a Hsp70. PFA0660w, a type II Hsp40, has been shown to be exported and possibly form a complex with PfHsp70-x in the infected erythrocyte cytosol. However, the chaperone properties of PFA0660w and its interaction with human and parasite Hsp70s are yet to be investigated. Recombinant PFA0660w was found to exist as a monomer in solution, and was able to significantly stimulate the ATPase activity of PfHsp70-x but not that of a second plasmodial Hsp70 (PfHsp70-1) or a human Hsp70 (HSPA1A), indicating a potential specific functional partnership with PfHsp70-x. Protein binding studies in the presence and absence of ATP suggested that the interaction of PFA0660w with PfHsp70-x most likely represented a co-chaperone/chaperone interaction. Also, PFA0660w alone produced a concentrationdependent suppression of rhodanese aggregation, demonstrating its chaperone properties. Overall, we have provided the first biochemical evidence for the possible role of PFA0660w as a chaperone and as co-chaperone of PfHsp70-x. We propose that these chaperones boost the chaperone power of the infected erythrocyte, enabling successful protein trafficking and folding, and thereby making a fundamental contribution to the pathology of malaria

The Involvement of SMILE/TMTC3 in Endoplasmic Reticulum Stress Response

The state of operational tolerance has been detected sporadically in some renal transplanted patients that stopped immunosuppressive drugs, demonstrating that allograft tolerance might exist in humans. Several years ago, a study by Brouard et al. identified a molecular signature of several genes that were significantly differentially expressed in the blood of such patients compared with patients with other clinical situations. The aim of the present study is to analyze the role of one of these molecules over-expressed in the blood of operationally tolerant patients, SMILE or TMTC3, a protein whose function is still unknown.We first confirmed that SMILE mRNA is differentially expressed in the blood of operationally tolerant patients with drug-free long term graft function compared to stable and rejecting patients. Using a yeast two-hybrid approach and a colocalization study by confocal microscopy we furthermore report an interaction of SMILE with PDIA3, a molecule resident in the endoplasmic reticulum (ER). In accordance with this observation, SMILE silencing in HeLa cells correlated with the modulation of several transcripts involved in proteolysis and a decrease in proteasome activity. Finally, SMILE silencing increased HeLa cell sensitivity to the proteasome inhibitor Bortezomib, a drug that induces ER stress via protein overload, and increased transcript expression of a stress response protein, XBP-1, in HeLa cells and keratinocytes.In this study we showed that SMILE is involved in the endoplasmic reticulum stress response, by modulating proteasome activity and XBP-1 transcript expression. This function of SMILE may influence immune cell behavior in the context of transplantation, and the analysis of endoplasmic reticulum stress in transplantation may reveal new pathways of regulation in long-term graft acceptance thereby increasing our understanding of tolerance

Transcript analysis of the extended hyp-operon in the cyanobacteria Nostoc sp. strain PCC 7120 and Nostoc punctiforme ATCC 29133

<p>Abstract</p> <p>Background</p> <p>Cyanobacteria harbor two [NiFe]-type hydrogenases consisting of a large and a small subunit, the Hup- and Hox-hydrogenase, respectively. Insertion of ligands and correct folding of nickel-iron hydrogenases require assistance of accessory maturation proteins (encoded by the <it>hyp</it>-genes). The intergenic region between the structural genes encoding the uptake hydrogenase (<it>hupSL</it>) and the accessory maturation proteins (<it>hyp </it>genes) in the cyanobacteria <it>Nostoc </it>PCC 7120 and <it>N. punctiforme </it>were analysed using molecular methods.</p> <p>Findings</p> <p>The five ORFs, located in between the uptake hydrogenase structural genes and the <it>hyp</it>-genes, can form a transcript with the <it>hyp</it>-genes. An identical genomic localization of these ORFs are found in other filamentous, N₂-fixing cyanobacterial strains. In <it>N. punctiforme </it>and <it>Nostoc </it>PCC 7120 the ORFs upstream of the <it>hyp</it>-genes showed similar transcript level profiles as <it>hupS </it>(hydrogenase structural gene), <it>nifD </it>(nitrogenase structural gene), <it>hypC </it>and <it>hypF </it>(accessory hydrogenase maturation genes) after nitrogen depletion. <it>In silico </it>analyzes showed that these ORFs in <it>N. punctiform</it>e harbor the same conserved regions as their homologues in <it>Nostoc </it>PCC 7120 and that they, like their homologues in <it>Nostoc </it>PCC 7120, can be transcribed together with the <it>hyp</it>-genes forming a larger extended <it>hyp-</it>operon. DNA binding studies showed interactions of the transcriptional regulators CalA and CalB to the promoter regions of the extended <it>hyp</it>-operon in <it>N. punctiforme </it>and <it>Nostoc </it>PCC 7120.</p> <p>Conclusions</p> <p>The five ORFs upstream of the <it>hyp</it>-genes in several filamentous N₂-fixing cyanobacteria have an identical genomic localization, in between the genes encoding the uptake hydrogenase and the maturation protein genes. In <it>N. punctiforme </it>and <it>Nostoc </it>PCC 7120 they are transcribed as one operon and may form transcripts together with the <it>hyp</it>-genes. The expression pattern of the five ORFs within the extended <it>hyp</it>-operon in both <it>Nostoc punctiforme </it>and <it>Nostoc </it>PCC 7120 is similar to the expression patterns of <it>hupS</it>, <it>nifD</it>, <it>hypF </it>and <it>hypC</it>. CalA, a known transcription factor, interacts with the promoter region between <it>hupSL </it>and the five ORFs in the extended <it>hyp</it>-operon in both <it>Nostoc </it>strains.</p

Analysis of Chaperone mRNA Expression in the Adult Mouse Brain by Meta Analysis of the Allen Brain Atlas

The pathology of many neurodegenerative diseases is characterized by the accumulation of misfolded and aggregated proteins in various cell types and regional substructures throughout the central and peripheral nervous systems. The accumulation of these aggregated proteins signals dysfunction of cellular protein homeostatic mechanisms such as the ubiquitin/proteasome system, autophagy, and the chaperone network. Although there are several published studies in which transcriptional profiling has been used to examine gene expression in various tissues, including tissues of neurodegenerative disease models, there has not been a report that focuses exclusively on expression of the chaperone network. In the present study, we used the Allen Brain Atlas online database to analyze chaperone expression levels. This database utilizes a quantitative in situ hybridization approach and provides data on 270 chaperone genes within many substructures of the adult mouse brain. We determined that 256 of these chaperone genes are expressed at some level. Surprisingly, relatively few genes, only 30, showed significant variations in levels of mRNA across different substructures of the brain. The greatest degree of variability was exhibited by genes of the DnaJ co-chaperone, Tetratricopeptide repeat, and the HSPH families. Our analysis provides a valuable resource towards determining how variations in chaperone gene expression may modulate the vulnerability of specific neuronal populations of mammalian brain

Plasmids and Rickettsial Evolution: Insight from Rickettsia felis

BACKGROUND: The genome sequence of Rickettsia felis revealed a number of rickettsial genetic anomalies that likely contribute not only to a large genome size relative to other rickettsiae, but also to phenotypic oddities that have confounded the categorization of R. felis as either typhus group (TG) or spotted fever group (SFG) rickettsiae. Most intriguing was the first report from rickettsiae of a conjugative plasmid (pRF) that contains 68 putative open reading frames, several of which are predicted to encode proteins with high similarity to conjugative machinery in other plasmid-containing bacteria. METHODOLOGY/PRINCIPAL FINDINGS: Using phylogeny estimation, we determined the mode of inheritance of pRF genes relative to conserved rickettsial chromosomal genes. Phylogenies of chromosomal genes were in agreement with other published rickettsial trees. However, phylogenies including pRF genes yielded different topologies and suggest a close relationship between pRF and ancestral group (AG) rickettsiae, including the recently completed genome of R. bellii str. RML369-C. This relatedness is further supported by the distribution of pRF genes across other rickettsiae, as 10 pRF genes (or inactive derivatives) also occur in AG (but not SFG) rickettsiae, with five of these genes characteristic of typical plasmids. Detailed characterization of pRF genes resulted in two novel findings: the identification of oriV and replication termination regions, and the likelihood that a second proposed plasmid, pRFδ, is an artifact of the original genome assembly. CONCLUSION/SIGNIFICANCE: Altogether, we propose a new rickettsial classification scheme with the addition of a fourth lineage, transitional group (TRG) rickettsiae, that is unique from TG and SFG rickettsiae and harbors genes from possible exchanges with AG rickettsiae via conjugation. We offer insight into the evolution of a plastic plasmid system in rickettsiae, including the role plasmids may have played in the acquirement of virulence traits in pathogenic strains, and the likely origin of plasmids within the rickettsial tree