Toward Better Understanding on How Group A <em>Streptococcus</em> Manipulates Human Fibrinolytic System

Group A Streptococcus pyogenes (GAS) is a human pathogen that commonly causes superficial infections such as pharyngitis, but can also lead to systemic and fatal diseases. GAS infection remains to be a major threat in regions with insufficient medical infrastructures, leading to half a million deaths annually worldwide. The pathogenesis of GAS is mediated by a number of virulence factors, which function to facilitate bacterial colonization, immune evasion, and deep tissue invasion. In this review, we will discuss the mechanism of molecular interaction between the host protein and virulence factors that target the fibrinolytic system, including streptokinase (SK), plasminogen-binding group A streptococcal M-like protein (PAM), and streptococcal inhibitor of complement (SIC). We will discuss our current understanding, through structural studies, on how these proteins manipulate the fibrinolytic system during infection

Juno: a Python-based graphical package for optical system design

This report introduces Juno, a modular Python package for optical design and simulation. Juno consists of a complete library that includes a graphical user interface to design and visualise arbitrary optical elements, set up wave propagation simulations and visualise their results. To ensure an efficient visualisation of the results, all simulation data are stored in a structured database that can filter and sort the output. Finally, we present a practical use case for Juno, where optical design and fabrication are interlaced in a feedback cycle. The presented data show how to compare the simulated and the measured propagation; if a difference or unexpected behaviour is found, we show how to convert and import the optical element profile from a profilometer measurement. The propagation through the profile can provide immediate feedback about the quality of the element and a measure of the effects brought by differences between the idealised and the actual profile, therefore, allowing to exclude the experimental errors and to weigh every aspect of fabrication errors.Comment: The software is available at https://github.com/DeMarcoLab/jun

The major human and mouse granzymes are structurally and functionally divergent

Approximately 2% of mammalian genes encode proteases. Comparative genomics reveals that those involved in immunity and reproduction show the most interspecies diversity and evidence of positive selection during evolution. This is particularly true of granzymes, the cytotoxic proteases of natural killer cells and CD8+ T cells. There are 5 granzyme genes in humans and 10 in mice, and it is suggested that granzymes evolve to meet species-specific immune challenge through gene duplication and more subtle alterations to substrate specificity. We show that mouse and human granzyme B have distinct structural and functional characteristics. Specifically, mouse granzyme B is 30 times less cytotoxic than human granzyme B and does not require Bid for killing but regains cytotoxicity on engineering of its active site cleft. We also show that mouse granzyme A is considerably more cytotoxic than human granzyme A. These results demonstrate that even “orthologous” granzymes have species-specific functions, having evolved in distinct environments that pose different challenges

Conformational Change in the Chromatin Remodelling Protein MENT

Chromatin condensation to heterochromatin is a mechanism essential for widespread suppression of gene transcription, and the means by which a chromatin-associated protein, MENT, induces a terminally differentiated state in cells. MENT, a protease inhibitor of the serpin superfamily, is able to undergo conformational change in order to effect enzyme inhibition. Here, we sought to investigate whether conformational change in MENT is ‘fine-tuned’ in the presence of a bound ligand in an analogous manner to other serpins, such as antithrombin where such movements are reflected by a change in intrinsic tryptophan fluorescence. Using this technique, MENT was found to undergo structural shifts in the presence of DNA packaged into nucleosomes, but not naked DNA. The contribution of the four Trp residues of MENT to the fluorescence change was mapped using deconvolution analysis of variants containing single Trp to Phe mutations. The analysis indicated that the overall emission spectra is dominated by a helix-H tryptophan, but this residue did not dominate the conformational change in the presence of chromatin, suggesting that other Trp residues contained in the A-sheet and RCL regions contribute to the conformational change. Mutagenesis revealed that the conformational change requires the presence of the DNA-binding ‘M-loop’ and D-helix of MENT, but is independent of the protease specificity determining ‘reactive centre loop’. The D-helix mutant of MENT, which is unable to condense chromatin, does not undergo a conformational change, despite being able to bind chromatin, indicating that the conformational change may contribute to chromatin condensation by the serpin

The REFOLD database: a tool for the optimization of protein expression and refolding

A large proportion of proteins expressed in Escherichia coli form inclusion bodies and thus require renaturation to attain a functional conformation for analysis. In this process, identifying and optimizing the refolding conditions and methodology is often rate limiting. In order to address this problem, we have developed REFOLD, a web-accessible relational database containing the published methods employed in the refolding of recombinant proteins. Currently, REFOLD contains >300 entries, which are heavily annotated such that the database can be searched via multiple parameters. We anticipate that REFOLD will continue to grow and eventually become a powerful tool for the optimization of protein renaturation. REFOLD is freely available at

RAB-Like 2 Has an Essential Role in Male Fertility, Sperm Intra-Flagellar Transport, and Tail Assembly

A significant percentage of young men are infertile and, for the majority, the underlying cause remains unknown. Male infertility is, however, frequently associated with defective sperm motility, wherein the sperm tail is a modified flagella/cilia. Conversely, a greater understanding of essential mechanisms involved in tail formation may offer contraceptive opportunities, or more broadly, therapeutic strategies for global cilia defects. Here we have identified Rab-like 2 (RABL2) as an essential requirement for sperm tail assembly and function. RABL2 is a member of a poorly characterized clade of the RAS GTPase superfamily. RABL2 is highly enriched within developing male germ cells, where it localizes to the mid-piece of the sperm tail. Lesser amounts of Rabl2 mRNA were observed in other tissues containing motile cilia. Using a co-immunoprecipitation approach and RABL2 affinity columns followed by immunochemistry, we demonstrated that within developing haploid germ cells RABL2 interacts with intra-flagella transport (IFT) proteins and delivers a specific set of effector (cargo) proteins, including key members of the glycolytic pathway, to the sperm tail. RABL2 binding to effector proteins is regulated by GTP. Perturbed RABL2 function, as exemplified by the Mot mouse line that contains a mutation in a critical protein-protein interaction domain, results in male sterility characterized by reduced sperm output, and sperm with aberrant motility and short tails. Our data demonstrate a novel function for the RABL protein family, an essential role for RABL2 in male fertility and a previously uncharacterised mechanism for protein delivery to the flagellum.This work was supported by grants from the NHMRC to MKO (#606445) and CJO, the Australian Research Council (MKO, RJA, and CJO), the New South Wales Cancer Council (CJO), Cancer Institute New South Wales (CJO), Banque Nationale de Paris-Paribas Australia and New Zealand (CJO), RT Hall Trust (CJO), and the National Breast Cancer Foundation (CJO). JCYL is the recipient of a NHMRC PhD scholarship. MKO and CJO are the recipients of NHMRC Senior Research Fellowships (#545805 and #481310). CCG is the recipient an NHMRC Australia Fellowship. JCW is the recipient of an Australian Research Council Federation Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

An ultrastructural investigation of tumors undergoing regression mediated by immunotherapy

While immunotherapy employing chimeric antigen receptor (CAR) T cells can be effective against a variety of tumor types, little is known about what happens within the tumor at an ultrastructural level during tumor regression. Here, we used transmission electron microscopy to investigate morphologic and cellular features of tumors responding to immunotherapy composed of adoptive transfer of dual-specific CAR T cells and a vaccine, supported by preconditioning irradiation and interleukin-2. Tumors responded rapidly, and large areas of cell death were apparent by 4 days after treatment. The pleomorphic and metabolically active nature of tumor cells and phagocytic activity of macrophages were apparent in electron microscopic images of tumors prior to treatment. Following treatment, morphologic features of various types of tumor cell death were observed, including apoptosis, paraptosis and necrosis. Large numbers of lipid droplets were evident in tumor cells undergoing apoptosis. Macrophages were the predominant leukocyte type infiltrating tumors before treatment. Macrophages decreased in frequency and number after treatment, whereas an increasing accumulation of neutrophils and T lymphocytes was observed following treatment. Phagocytic activity of macrophages and neutrophils was apparent, while T cells could be observed in close association with tumor cells with potential immunological synapses present. These observations highlight the cellular composition and ultrastructural appearance of tumors undergoing regression mediated by immunotherapy

Efficient large-scale protein sequence comparison and gene matching to identify orthologs and co-orthologs

Broadly, computational approaches for ortholog assignment is a three steps process: (i) identify all putative homologs between the genomes, (ii) identify gene anchors and (iii) link anchors to identify best gene matches given their order and context. In this article, we engineer two methods to improve two important aspects of this pipeline [specifically steps (ii) and (iii)]. First, computing sequence similarity data [step (i)] is a computationally intensive task for large sequence sets, creating a bottleneck in the ortholog assignment pipeline. We have designed a fast and highly scalable sort-join method (afree) based on k-mer counts to rapidly compare all pairs of sequences in a large protein sequence set to identify putative homologs. Second, availability of complex genomes containing large gene families with prevalence of complex evolutionary events, such as duplications, has made the task of assigning orthologs and co-orthologs difficult. Here, we have developed an iterative graph matching strategy where at each iteration the best gene assignments are identified resulting in a set of orthologs and co-orthologs. We find that the afree algorithm is faster than existing methods and maintains high accuracy in identifying similar genes. The iterative graph matching strategy also showed high accuracy in identifying complex gene relationships. Standalone afree available from http://vbc.med.monash.edu.au/∼kmahmood/afree. EGM2, complete ortholog assignment pipeline (including afree and the iterative graph matching method) available from http://vbc.med.monash.edu.au/∼kmahmood/EGM2