93 research outputs found

    Dissecting Extracellular Matrix Internalisation Mechanisms using Functional Genomics

    Get PDF
    Breast and ovarian malignancies account for one third of female cancers. The role of the stroma in supporting invasive growth in breast cancer has become clear. Breast cancer cells interact and respond to the cues from the surrounding extracellular matrix (ECM). Integrins are main cell adhesion receptors and key players in invasive migration by linking the ECM to the actin cytoskeleton. In addition, integrins mediate distinctive biochemical and biomechanical signals to support cancer invasion. The role of matrix proteases in promoting ECM degradation and cancer dissemination has been extensively studied; however, cancer cells possess additional means to support those processes, such as integrin-mediated ECM endocytosis and consequent degradation in the lysosomes. Internalisation of the extracellular matrix is upregulated in invasive breast cancer. Nonetheless, the mechanisms by which cancer cells regulate this process are poorly understood. We developed a high throughput pH sensitive system to detect ECM uptake. Here, we show that MDA-MB-231 breast cancer cells converge in macropinocytosis to internalise diverse ECM components and we confirm that this process is modulated by PAK1. To unravel which ECM components breast cancer cells internalise in a complex environment (namely, cell derived matrices), we performed mass spectrometry. Proteomic analysis identified Annexin A6, Collagen VI, Tenascin C and fibronectin, among other matrisome proteins, to be internalised by invasive breast cancer cells. Following ECM endocytosis, ECM is targeted for lysosomal degradation. To unravel the molecular mechanisms behind this process, we performed a trafficking screen and identified the AP3 complex, VAMP7, Arf1 and ARFGEF2. Our results suggest that the AP3 complex may regulate ECM-integrin delivery to lysosomes. To gain more insight on the signalling pathways governing macropinocytosis in breast cancer cells, we performed a kinase and phosphatase screen that unravelled MAP3K1 and PPP2R1A, a subunit of protein phosphatase 2A (PP2A) as relevant regulators of ECM endocytosis. Furthermore, our data suggests that p38 mitogen-activated protein kinase (MAPK) activation upon binding to the ECM is required for ECM macropinocytosis. Outstandingly, inhibiting p38 MAPK led to profound changes in the ability of breast cancer cells to migrate in cell derived matrices. Previous work from the Rainero lab focused on characterising the receptors involved in ECM internalisation; α2ÎČ1 integrin was identified as the main regulator of ECM uptake in MDA-MB-231 cells. In particular, α2ÎČ1 integrin has been shown to activate p38 MAPK pathway. Taken together, we hypothesise that binding of ECM to α2ÎČ1 integrin results in the activation of PAK1 and MAP3K1, which in turn leads to ECM endocytosis. p38 MAPK activity may induce changes in actin polymerisation via PPP2R1A and/or focal adhesion turnover, which consequently promotes ECM macropinocytosis and invasive migration

    Exploring Humoral Immune Responses by Mass Spectrometry: Resolving Structures, Interactions, and Clonal Repertoires of Antibodies

    Get PDF
    In his thesis “Exploring Humoral Immune Responses by Mass Spectrometry”, Maurits den Boer uses mass spectrometry to shed new light on antibody responses. Antibodies play a crucial role in the immune protection against threats like bacteria, viruses, and cancers. When valuable antibodies are discovered, they can therefore be reproduced for use as a medicine. A better understanding of their structures, interactions, and repertoires is therefore key to finding novel treatments for many diseases. In the first part of his thesis, Maurits and coworkers used mass spectrometry to study antibody structures and interactions, leading to two major findings. They first uncovered a mechanism by which Staphylococcus aureus bacteria can evade antibody responses, and how this mechanism may be circumvented in future therapies. Second, he redefined the textbook structure of circulating IgM antibodies by showing that they are universally attached to an extra protein. This may have major implications for how these antibodies function, and their use as therapeutics. In a second line of research, Maurits focused on the development of innovative techniques for antibody repertoire analysis and discovery. Together with coworkers, he explored the use of electron-based fragmentation mass spectrometry, developing methods to obtain valuable pieces of antibody sequence information. Finally, he combined multiple layers of mass spectrometry analysis to discover and fully determine the sequence of a malignant patient antibody. Combined, this demonstrates the promise of mass spectrometry as a compelling new approach for therapeutic antibody discovery

    Chapter 34 - Biocompatibility of nanocellulose: Emerging biomedical applications

    Get PDF
    Nanocellulose already proved to be a highly relevant material for biomedical applications, ensued by its outstanding mechanical properties and, more importantly, its biocompatibility. Nevertheless, despite their previous intensive research, a notable number of emerging applications are still being developed. Interestingly, this drive is not solely based on the nanocellulose features, but also heavily dependent on sustainability. The three core nanocelluloses encompass cellulose nanocrystals (CNCs), cellulose nanofibrils (CNFs), and bacterial nanocellulose (BNC). All these different types of nanocellulose display highly interesting biomedical properties per se, after modification and when used in composite formulations. Novel applications that use nanocellulose includewell-known areas, namely, wound dressings, implants, indwelling medical devices, scaffolds, and novel printed scaffolds. Their cytotoxicity and biocompatibility using recent methodologies are thoroughly analyzed to reinforce their near future applicability. By analyzing the pristine core nanocellulose, none display cytotoxicity. However, CNF has the highest potential to fail long-term biocompatibility since it tends to trigger inflammation. On the other hand, neverdried BNC displays a remarkable biocompatibility. Despite this, all nanocelluloses clearly represent a flag bearer of future superior biomaterials, being elite materials in the urgent replacement of our petrochemical dependence

    Insertion and interaction of transmembrane helices, from basic principles to rational design

    Get PDF
    The main objective of this thesis was to study α-helical membrane protein segments, from basic principles to rational design. Our intention was to investigate a wide spectrum of functions developed by the TMDs far beyond from its structural role anchoring proteins to membranes. The final goal of this thesis is to delve deep in our understanding of membrane protein biogenesis from insertion and topology determination to better characterize these ‘greasy’ proteins. In particular, we wanted to make progress on depicting how α-helical TMD-TMD interactions work and their importance in the regulation of different cellular processes like apoptosis and the control of cellular death. A better understanding of these regulation processes can improve our current knowledge and lead to new therapeutic targets. In this direction, we ought to explore the inhibition of TMD-TMD interactions as a target to regulate cell death. The results of this thesis were obtained using some of the more relevant techniques from the biochemistry and molecular biology toolbox as: plasmid cloning, site-directed mutagenesis, SDS-PAGE electrophoresis, western blotting, transcription/translation expression in vitro, apoptosis assays in cell lines (HeLa), and also protein engineering approaches based on bimolecular complementation protocols like BiFC (Bimolecular Fluorescence Complementation) and BLaTM (based on the use of a split ÎČ-Lactamase fused to a TMD). These techniques led us to a better description of how polar residues can be inserted into the membrane. They also helped us to improve our understanding of the insertion process, the topology determination of viral proteins and to better comprehend the complexity of TMD-TMD interactions and their role to fine-tune the apoptotic networks, pointing out the importance of the TM region. Finally, we used these interactions to computationally design TM inhibitors as a new possible therapeutic agents to regulate cellular death by modulating TMD-TMD interactions. The research related to this work resulted in four first-author publications and one manuscript that is currently being revised, which constitute the four chapters of this thesis, and two more papers included in the annexed section. The annexed papers (see section 8), whereof I am the first author, include a review depicting some of the most relevant methods used in this thesis to study TMD interactions. Additionally, a patent (P202230029) including part of this work has been filled by the University of Valencia. The following pages are a summary of the results and discussion of these papers organized in four chapters. The objective of this work was to estimate the energetic contribution of intra-helical salt bridges to the insertion of TMDs into biological membranes. The presence of intra-helical salt bridges in TMDs, as well as their impact on insertion, has not been properly studied yet. α-helical TMDs are largely composed of apolar residues because of the hydrophobic nature of the membrane. Nevertheless, in some cases, membrane embedded proteins carry polar amino acids in their TMDs for proper folding or functional purposes (Baeza-Delgado et al., 2013). In fact, the presence of polar and charged amino acids in TMDs is more frequent than what would be expected according to the hydrophobic nature of the environment (Bañó-Polo et al., 2012), especially when these residues are present in pairs. Salt bridges are electrostatic interactions between negatively and positively charged amino acids that play a prevalent role in protein stabilization (Marqusee and Baldwin, 1987). Analysing the TMDs from membrane protein structures we observed that charged pairs of amino acids are especially prevalent at positions i, i+1; i, i+3 and i, i+4. Oppositely paired charges located at these positions could potentially form salt bridges as they are all on the same face of the helix and are close enough, in terms of atomic distances. To assess the contribution of putative salt bridges to the translocon-mediated membrane insertion, we used as a vehicle the leader peptidase (Lep) protein from Escherichia coli. The Lep protein consists of two TMDs (H1 and H2) connected by a cytosolic loop (P1) and a large C-terminal (P2) domain, which inserts into endoplasmic reticulum (ER)-derived rough microsomes (RM) with both termini located in the lumen of the microsomes (von Heijne, 1989). The designed TMDs were inserted into the luminal P2 domain and flanked by two N-linked glycosylation acceptor sites (G1 and G2). Glycosylation occurs exclusively in the lumen of the ER (or the microsomes) because of the location of the oligosaccharyltransferase (OST) active site (a translocon-associated enzyme responsible for the oligosaccharide transfer) (Braunger et al., 2018). Glycosylation of an acceptor site increases the apparent molecular mass of the protein (~2.5 kDa), which facilitates its identification by gel electrophoresis. We first compared the effect of the oppositely charged Lys and Asp residues on the insertion of a 19-residue-long hydrophobic artificial scaffold (L4/A15, 4 leucines and 15 alanines), designed to stably insert into the RM membranes and “insulated” from the surrounding sequence by N- and C-terminal GGPG- and -GPGG (G, glycin; P, prolin) tetrapeptides. Single Lys and Asp residues were placed at positions 8 and 12 respectively, and pairs of Lys-Asp residues were designed to cover positions 7–12 (more than one helical turn). When pairs of charged residues were present, our results showed a tendency to insert more efficiently when oppositely paired charges were placed at positions that are permissive distances for salt bridge formation (i, i+1; i, i+3; i, i+4), an effect not observed in the current prediction algorithms. Similar results were obtained on a different Leu/Ala background with a slightly higher insertion efficiency (L5/A14, 5 leucines and 14 alanines). After testing the effect of oppositely charged residues in the insertion of model sequences, we decided to look for salt bridges in natural proteins. We analysed the TMDs from high-resolution membrane protein structures. Then, we generated a list of potential candidates for further in vitro and whole cell studies. We selected the helix G from Natronomonas pharaonis halorhodopsin (PDB code: 3QBG) and the helix A from the Oryctolagus cuniculus calcium ATPase (PDB code: 1SU4). In both cases we studied the insertion of these helices using in vitro assays (Lep in microsomes) and eukaryotic cells (Lep and CLTM systems). Halorhodopsin from Natronomonas pharaonis is a protein made up of seven TMDs (A to G) and a retinal chromophore bound via a protonated Schiff base to the Δ-amino group of the Lys258, located in the middle of helix G (Kanada et al., 2011). In silico analysis of the apoprotein 3QBG structure showed a Lys-Arg pair, involving i, i+4 Lys258 and Asp254 residues from helix G. The distance between charges in the crystal structure of the apoprotein was about 3.5 Å, a permissive distance for a salt bridge formation. Then, we designed three mutants that were supposed to perturb salt bridge interaction in different ways: the K258D mutant places two charged residues with the same polarity at positions i, i+4; the K258A mutant replaces one of the charged residues by a non-polar amino acid, and the K258Y/Y259K double mutant places the two different charges at a non-permissive position for salt bridge formation (i, i+5) while preserving amino acid composition. The results of the Lep-based glycosylation assay indicated that wild type and K258A mutant are inserted properly, but when the salt bridge is disrupted, insertion efficiency decreased substantially (~0.5 kcal/mol). These results were replicated in HEK-293T cells. Cells transfected with the chimera containing helix G native sequence rendered almost full insertion. In contrast, cells transfected with the construct harbouring i, i+5 sequence rendered almost exclusively membrane translocation. These results emphasized the relevance of intra-helical salt bridges in translocon-mediated TM insertion, especially in the cellular environment. Our second studied protein was the calcium ATPase from Oryctolagus cuniculus. This protein contains a bundle of 10 TM helices (A to J) (Toyoshima et al., 2000). In silico analysis of 1SU4 highlighted an Asp-Arg pair, involving Asp59 and Arg63, in the centre of helix A. The distance between these charges in the crystal structure was about 3.0 Å, clearly within the permissive range for salt bridge formation. Again, the results of the Lep-based glycosylation assay demonstrated the efficient insertion of the native helix A sequence. Importantly, when the paired residues were placed at the non-permissive i, i+5 positions the insertion efficiency was decreased, with a ΔGapp estimated in ~0.7 kcal/mol. Similar results were also observed in HEK-293T cell experiments. In summary, charged residues found in α-helices can be important for function (Lin and Lin, 2018) but also provide a stabilizing effect (Armstrong and Baldwin, 1993). Pairs of oppositely charged residues can form salt bridges that could allow them to facilitate TMD insertion when facing the hydrophobic environment (Walther and Ulrich, 2014). Salt bridges might also be important for shielding the charges during translocon-mediated TMD insertion (Whitley et al., 2021). In TMDs, oppositely charged residue pairs are more prevalent at positions i, i+1; i, i+3 and i, i+4, compatible with salt-bridge formation. By analysing two native helices containing intra-helical salt bridges we found that the free energy of insertion (ΔGapp) is significantly reduced when both oppositely charged residues are spaced at a permissive distance. These results indicate that intra-helix salt bridges could form during translocon-assisted insertion or even earlier, since TM helices can be compacted inside the ribosome exit tunnel (Bañó-Polo et al., 2018). The reduction of ΔGapp in these natural proteins is between 0.5-0.7 kcal/mol. As found in the case of the halorhodopsin helix G, this reduction might be higher in the cell context, since some auxiliary components of the membrane insertion machinery (Chitwood and Hegde, 2020; Shurtleff et al., 2018; Tamborero et al., 2011) might be not represented in the microsomal vesicle preparations. Current prediction algorithms for membrane protein insertion tend to overestimate the free energy penalty of charged residues in TMDs. Incorporating the effect of potential salt bridges in the reduction of ΔGapp during membrane integration could help to improve future prediction tools. As soon as the genome of the SARS-CoV-2, the virus responsible of the COVID-19 pandemic, was released, we started a project aimed to study the envelope (E) protein. The E protein is the smallest and has the lowest copy number among the membrane proteins found in the lipid envelope of mature virus particles (Bar-On et al., 2020). However, it is critical for pathogenesis of the SARS-CoV-2 and other human coronaviruses (AlmazĂĄn et al., 2013; Ruch and Machamer, 2012; Xia et al., 2021; Zheng et al., 2021) and has been described as a viroporin. Interestingly, the sgRNA encoding E protein is one of the most abundantly expressed transcripts despite the protein having a low copy number in mature viruses (Wu et al., 2020). This sgRNA encodes a 75 residues long polypeptide with a predicted molecular weight of approximately 8 kDa. Comparative sequence analysis of the E protein of SARS-CoV-2 and the other six known human coronaviruses do not reveal any large homologous/identical regions, with only the initial Met, Leu39, Cys40 and Pro54 being ubiquitously conserved. Regarding overall sequence similarity SARS-CoV-2 E protein has the highest similarity to SARS-CoV (94.74%) with only minor differences, followed by MERS-CoV (36.00%). Interestingly, sequence similarities are significantly lower for the other four human coronaviruses, which usually cause mild to moderate upper-respiratory tract illness typical for common cold. To determine its membrane topology, we assayed E protein insertion in microsomal membranes using in vitro transcription/translation experiments in the presence of [35S]-labelled amino acids but also in eukaryotic membranes using HEK-293T cells. Using a glycosylation based assay as a molecular reporter, we determined that the SARS-CoV-2 E protein integrates into the membrane co-translationally as a single-spanning membrane protein with an Ntlum/Ctcyt orientation in in vitro and in vivo systems. This topology is compatible with the ion channel capacity described previously (VerdiĂĄ-BĂĄguena et al., 2012). Furthermore, this topology is reinforced by different topological determinants present in the SARS-CoV-2 E protein sequence. In all seven human coronaviruses there is a strongly conserved positively charged residue placed after the hydrophobic region. It is worth to mention that this residue is an arginine (Arg38) in MERS-CoV, SARS-CoV and SARS-CoV-2, while in the other human coronaviruses is a lysine. Also, the alignment of MERS-CoV, SARS-CoV and SARS-CoV-2 E proteins unveils a tendency to accumulate a net positive charge balance C-terminally to the TMD, which correlates with the positive-inside rule, suggesting an increasing robustness in the topology determination from MERS-CoV to SARS-CoV-2. We experimentally confirmed this increasing robustness by modifying the full protein charge balance for all three pathogenic E proteins. In all three cases, the conserved Arg38 residue plays a limited role in the topology determination. Our data also suggested that the Arg to Glu mutation present in both SARS-CoVs’ N-terminus compared with MERS-CoV sequence, is most likely one of the mechanisms contributing to the proved topology robustness of the SARS-CoVs by converting the net charge of 0 at N-terminal region of MERS-CoV into a -2 in both SARS-CoVs, in good agreement with the so-called “negative outside enrichment” rule (Baker et al., 2017). Programmed cell death is a fundamental process in the development of multicellular organisms contributing to the balance among cell death, proliferation, and differentiation, that is crucial for tissue development and homeostasis (Kerr et al., 1972). Also, protection and defence against many disorders, including cancer and pathogen-related diseases, rely on apoptosis to eliminate the affected cells (HĂ€cker, 2018; Hua et al., 2019). One of the primary modulators of apoptosis is the B-cell lymphoma 2 (Bcl2) protein family (Kim et al., 2006). The proteins in this family can be divided in anti-apoptotic (e.g., Bcl2 and BclxL) (Boise et al., 1993), pro-apoptotic (e.g., Bax and Bak)(Oltvai et al., 1993), and BH3-only apoptosis activators (e.g., Bid and Bmf) (Wang et al., 1996). Most pro-apoptotic and anti-apoptotic proteins in this family share up to four main Bcl2 sequence homology domains, known as BH1, BH2, BH3, and BH4, while BH3-only members have solely the BH3 domain. In addition, many Bcl2 family members have a TMD in the carboxyl-terminal end that effectively allows for insertion of the protein into the target lipid bilayer (Delbridge et al., 2016). Cellular Bcl2 (cBcl2) proteins can physically interact with each other, forming homo-and hetero-oligomers that are crucial for programmed cell death regulation (Cosentino and GarcĂ­a-SĂĄez, 2017; Kelekar et al., 1997; Oltvai et al., 1993; O’Neill et al., 2006; Wang et al., 1996; Xie et al., 1998). To prevent the premature death of host cells, viruses have developed functional homologues of cBcl2, known as viral Bcl2 (vBcl2), as a strategy to modulate cell death (Kvansakul et al., 2017; Polčic et al., 2017). Although there is low sequence homology between vBcl2 and cBcl2, crystal structures reveal a structural homology in key domains (Galluzzi et al., 2008; Kvansakul and Hinds, 2013). In this work, we firstly proved that vBcl2 contain a functional TMD in the Ct end, like their cellular counterparts. We selected six vBcl2 proteins from two distinct viral families (3 hepesviruses and 3 poxviruses). BHRF1 (Human gammaherpesvirus 4 – Epstein–Barr virus, HHV4) (Pearson et al., 1987), ORF16 (Human gammaherpesvirus 8 – Kaposi’s sarcoma–associated herpesvirus, HHV8) (Cheng et al., 1997), ORF16 (Bovine gammaherpesvirus 4, BoHV4) (Bellows et al., 2000), F1L (Vaccinia virus, VacV) (Nichols et al., 2017; Wasilenko et al., 2003), M11L (Myxoma virus, MyxV) (Douglas et al., 2007; Nichols et al., 2017), and ORFV125 (Orf virus, OrfV) (Westphal et al., 2007). To avoid confusion, here we use the viral acronym to refer to the vBcl2 protein. In silico analyses suggested the presence of TMDs in the selected vBcl2. Then, we aimed to explore the membrane insertion capacity of the predicted segments using an in vitro assay based on the E. coli leader peptidase (Lep) described before (see section 3.1). Using this glycosylation-based system we determined that all the studied vBcl2 regions were efficiently inserted into microsome membranes. After determining that the vBcl2s included a Ct end anchoring TMD, we sought to study whether their role went beyond that of a structural anchor. As interactions between cBcl2 TMDs have been reported in biological membranes (Andreu-FernĂĄndez et al., 2017), we decided to study the vBcl2 TMD capability of homo and hetero-oligomerization in biological membranes. For this purpose, we used two different systems of bimolecular complementation based on different reporters and in different model organisms. We used BiFC (bimolecular fluorescent complementation) approach (Kerppola, 2006) adapted for the study of intramembrane interactions in eukaryotic cells (Andreu-FernĂĄndez et al., 2017; Grau et al., 2017). This technique is based on a split venus fluorescent protein (VFP). Each of the two non-fluorescent fragments of the VFP is fused to one of the studied TMDs and expressed in eukaryotic cells. The two fragments of the VFP have no affinity for each other. The VFP will be reconstituted (and its fluorescence) only when TMD-TMD interaction is reported. The TMDs of HHV4, HHV8, VacV, MyxV, and OrfV showed a homo-interaction capability above the controls and similar to that observed with cBcl2 TMD. However, BoHV did not show VFP-associated ïŹ‚uorescence significantly higher than the negative controls. Western blot analysis showed comparable expression levels of all chimeras. To investigate the potential TMD-TMD heteromeric interactions between vBcl2 and cBcl2 we used the previously described BiFC approach. We investigated the potential TMD-TMD interactions between vBcl2 and the anti-apoptotic cellular Bcl2 and BclxL proteins. Interestingly, all viral TMDs included in the assay could interact with the TMD of Bcl2. However, although the majority of vBcl2 TMDs also interacted with BclxL TMD, BoHV and MyxV TMDs did not. We also analysed the interactions between vBcl2 TMDs and the TMDs of the cellular pro-apoptotic Bax and Bak proteins. In the absence of apoptotic stimulus and independent of any cytosolic (soluble) domain contacts, all three poxviral TMDs (VacV, MyxV, and OrfV) could interact with Bax and Bak TMDs. On the contrary, herpesviruses HHV4, HHV8, and BoHV TMDs showed no interaction with Bax or Bak TMDs. Finally, we included in the BiFC-based screening the TMDs from BH3-only apoptotic modulators Bik and Bmf (Andreu-FernĂĄndez et al., 2016). The TMDs of HHV4, HHV8, VacV, and OrfV could interact with the TMD of Bik. However, viral interactions with the TMD of Bmf were limited, only the HHV8 TMD interacts with Bmf TMD. Also, we further explored some of these interactions (e.g., HHV8-Bcl2 and MyxV-Bax) using the BLaTM assay (Schanzenbach et al., 2017) and computational modelling. BLaTM is a genetic tool designed to study TMD–TMD interactions in bacterial membranes. This tool is based on the use of a split ÎČ-Lactamase fused to the TMDs of study and expressed in E. coli cells. The TMD-TMD interaction aims the reconstitution of the ÎČ-Lactamase activity and is reported by ampicillin resistance. In this assay, the LD50 of the antibiotic served as an indicator of the interaction strength. Of note, bacteria can grow only in the presence of ampicillin when the ÎČ-lactamase is reconstituted in the periplasm. Therefore, the BLaTM assay also reports the insertion of the tested regions. With this system we double checked some of the previously studied interactions in a more quantitative manner. Overall, our findings point out an intricate network of interactions between the TMDs of viral and cellular origin. Finally, we wanted to determine if the TMD-TMD interaction network described played a role in apoptosis modulation. To that end, we transfected HeLa cells with Bcl2, HHV8, or MyxV either with or without the TMD (FL and ΔTMD, respectively). We also included chimeras in which the TMD of each of the previously described proteins was replaced by the TMD of the non-apoptotic mitochondrial protein TOMM20 (Bcl2-T20, HHV8-T20, and MyxV-T20, respectively), which our earlier studies suggested could not interact with any viral or cellular Bcl2 TMD. Cells were transfected with the appropriate constructs and then treated with doxorubicin to induce apoptosis (Rooswinkel et al., 2014). As expected, the FL proteins prevented apoptosis. When the TMD was removed, however, none of the proteins retained their anti-apoptotic effect. Similarly, the chimeras carrying the TMD of T20 could not control the doxorubicin-induced apoptosis, although localizing in the same membranes as the FL protein. These experiments were also replicated using different apoptotic stimulus as viral-induced apoptosis or Bax-induced apoptosis (only for Bcl2 and MyxV; FL and T20 variants). In summary, we

    Antimicrobial Resistance at the Human-Animal Interface

    Get PDF
    Livestock-associated Methicillin-resistant Staphylococcus aureus (MRSA) are an emerging public-health issue in Australia, particularly amongst livestock and animal workers. We examined MRSA, isolated from humans and animals in Australia whole-genome sequencing and identified zoonotic and anthropozoonotic MRSA transmission, and antimicrobial-resistance gene transfer between MRSA of different host origin. This work highlights the need for expanded monitoring of microbial livestock pathogens and indicates the importance of prudent antimicrobial use in animal health

    Drug development progress in duchenne muscular dystrophy

    Get PDF
    Duchenne muscular dystrophy (DMD) is a severe, progressive, and incurable X-linked disorder caused by mutations in the dystrophin gene. Patients with DMD have an absence of functional dystrophin protein, which results in chronic damage of muscle fibers during contraction, thus leading to deterioration of muscle quality and loss of muscle mass over time. Although there is currently no cure for DMD, improvements in treatment care and management could delay disease progression and improve quality of life, thereby prolonging life expectancy for these patients. Furthermore, active research efforts are ongoing to develop therapeutic strategies that target dystrophin deficiency, such as gene replacement therapies, exon skipping, and readthrough therapy, as well as strategies that target secondary pathology of DMD, such as novel anti-inflammatory compounds, myostatin inhibitors, and cardioprotective compounds. Furthermore, longitudinal modeling approaches have been used to characterize the progression of MRI and functional endpoints for predictive purposes to inform Go/No Go decisions in drug development. This review showcases approved drugs or drug candidates along their development paths and also provides information on primary endpoints and enrollment size of Ph2/3 and Ph3 trials in the DMD space

    Cutaneous Melanoma Classification: The Importance of High-Throughput Genomic Technologies

    Get PDF
    Cutaneous melanoma is an aggressive tumor responsible for 90% of mortality related to skin cancer. In the recent years, the discovery of driving mutations in melanoma has led to better treatment approaches. The last decade has seen a genomic revolution in the field of cancer. Such genomic revolution has led to the production of an unprecedented mole of data. High-throughput genomic technologies have facilitated the genomic, transcriptomic and epigenomic profiling of several cancers, including melanoma. Nevertheless, there are a number of newer genomic technologies that have not yet been employed in large studies. In this article we describe the current classification of cutaneous melanoma, we review the current knowledge of the main genetic alterations of cutaneous melanoma and their related impact on targeted therapies, and we describe the most recent highthroughput genomic technologies, highlighting their advantages and disadvantages. We hope that the current review will also help scientists to identify the most suitable technology to address melanoma-related relevant questions. The translation of this knowledge and all actual advancements into the clinical practice will be helpful in better defining the different molecular subsets of melanoma patients and provide new tools to address relevant questions on disease management. Genomic technologies might indeed allow to better predict the biological - and, subsequently, clinical - behavior for each subset of melanoma patients as well as to even identify all molecular changes in tumor cell populations during disease evolution toward a real achievement of a personalized medicine

    Protein Structure

    Get PDF
    Since the dawn of recorded history, and probably even before, men and women have been grasping at the mechanisms by which they themselves exist. Only relatively recently, did this grasp yield anything of substance, and only within the last several decades did the proteins play a pivotal role in this existence. In this expose on the topic of protein structure some of the current issues in this scientific field are discussed. The aim is that a non-expert can gain some appreciation for the intricacies involved, and in the current state of affairs. The expert meanwhile, we hope, can gain a deeper understanding of the topic

    Artificial assembly of the HIV-1 capsid using DNA scaffolds

    Full text link
    The HIV-1 viral capsid is a conical-shaped protein shell consisting of approximately 1500 copies of a single capsid (CA) protein, arranged into a lattice of hexamers and precisely 12 pentamers. These pentamers form the vertices that result in the enclosed conical shape. The capsid encapsulates the viral genome and its associated replication machinery (known collectively as the capsid core), and has a functional role in almost every step of the early stage of the virus life cycle, including nuclear translocation, reverse transcription, and nuclear import, before undergoing controlled disassembly inside the nucleus to release the genomic content. The dynamics which control the coordinated behaviour of the capsid subunits are complex and not well understood, yet they remain critical to our understanding of the fundamental principles underlying viral self-assembly. Methods were established to create CA binding sites to specific regions of the DNA scaffolds to provide control over relative spatial positions of multiple subunits at low nanometre resolution. With this system, we were able to probe and measure CA inter-subunit binding kinetics for the first time using surface plasmon resonance. The approach provided sufficient sensitivity to measure extremely weak interactions between CA subunits via the intra-hexamer interface, and showed that binding of the sixth subunit within a hexamer may exhibit weak cooperativity. We also demonstrated that the inter-hexamer interface can be disrupted with a single point mutation. In addition, we structurally characterised the solution structure of the isolated CA hexamer using small-angle X-ray scattering and cryo-electron microscopy, which revealed close structural similarities with existing crystal structures and lattice-derived reconstructions of CA hexamers. We also used DNA scaffolds to assemble stable isolated hexamers without requiring salt-induced lattice assembly for the first time, and used this approach to attempt construction of a stable isolated CA pentamer in its native conformation. In this study, we present a new method for probing specific CA interactions within dimeric, hexameric and pentameric complexes, providing insight into the underlying kinetics of viral self-assembly. Furthermore, we demonstrate a bottom-up approach to assembly of stable CA structures using DNA nanostructures and discuss the potential of using DNA for assembling previously inaccessible, and larger capsid complexes
    • 

    corecore