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Pancreatic ductal adenocarcinoma (PDAC) is characterized by increasing fibrosis, which can enhance tumor progression and spread. Here, we undertook an unbiased temporal assessment of the matrisome of the highly metastatic KPC ( Pdx1-Cre , LSL-Kras G12D/+ , LSL-Trp53 R172H/+ ) and poorly metastatic KP fl C ( Pdx1-Cre , LSL-Kras G12D/+ , Trp53 fl/+ ) genetically engineered mouse models of pancreatic cancer using mass spectrometry proteomics. Our assessment at early-, mid-, and late-stage disease reveals an increased abundance of nidogen-2 (NID2) in the KPC model compared to KP fl C, with further validation showing that NID2 is primarily expressed by cancer-associated fibroblasts (CAFs). Using biomechanical assessments, second harmonic generation imaging, and birefringence analysis, we show that NID2 reduction by CRISPR interference (CRISPRi) in CAFs reduces stiffness and matrix remodeling in three-dimensional models, leading to impaired cancer cell invasion. Intravital imaging revealed improved vascular patency in live NID2-depleted tumors, with enhanced response to gemcitabine/Abraxane. In orthotopic models, NID2 CRISPRi tumors had less liver metastasis and increased survival, highlighting NID2 as a potential PDAC cotarget.

Small non-coding RNA or microRNA (miRNA) are critical regulators of eukaryotic cells. Dysregulation of miRNA expression and function has been linked to a variety of diseases including cancer. They play a complex role in cancers, having both tumour suppressor and promoter properties. In addition, a single miRNA can be involved in regulating several mRNAs or many miRNAs can regulate a single mRNA, therefore assessing these roles is essential to a better understanding in cancer initiation and development. Pancreatic cancer is a leading cause of cancer death worldwide, in part due to the lack of diagnostic tools and limited treatment options. The most common form of pancreatic cancer, pancreatic ductal adenocarcinoma (PDAC), is characterised by major genetic mutations that drive cancer initiation and progression. The regulation or interaction of miRNAs with these cancer driving mutations suggests a strong link between the two. Understanding this link between miRNA and PDAC progression may give rise to novel treatments or diagnostic tools. This review summarises the role of miRNAs in PDAC, the downstream signalling pathways that they play a role in, how these are being used and studied as therapeutic targets as well as prognostic/diagnostic tools to improve the clinical outcome of PDAC., (© 2024. The Author(s).)

In recent decades, the role of tumor biomechanics on cancer cell behavior at the primary site has been increasingly appreciated. However, the effect of primary tumor biomechanics on the latter stages of the metastatic cascade, such as metastatic seeding of secondary sites and outgrowth remains underappreciated. This work sought to address this in the context of triple negative breast cancer (TNBC), a cancer type known to aggressively disseminate at all stages of disease progression. Using mechanically tuneable model systems, mimicking the range of stiffness's typically found within breast tumors, it is found that, contrary to expectations, cancer cells exposed to softer microenvironments are more able to colonize secondary tissues. It is shown that heightened cell survival is driven by enhanced metabolism of fatty acids within TNBC cells exposed to softer microenvironments. It is demonstrated that uncoupling cellular mechanosensing through integrin β1 blocking antibody effectively causes stiff primed TNBC cells to behave like their soft counterparts, both in vitro and in vivo. This work is the first to show that softer tumor microenvironments may be contributing to changes in disease outcome by imprinting on TNBC cells a greater metabolic flexibility and conferring discrete cell survival advantages., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

The lysyl oxidase family represents a promising target in stromal targeting of solid tumors due to the importance of this family in crosslinking and stabilizing fibrillar collagens and its known role in tumor desmoplasia. Using small-molecule drug-design approaches, we generated and validated PXS-5505, a first-in-class highly selective and potent pan-lysyl oxidase inhibitor. We demonstrate in vitro and in vivo that pan-lysyl oxidase inhibition decreases chemotherapy-induced pancreatic tumor desmoplasia and stiffness, reduces cancer cell invasion and metastasis, improves tumor perfusion and enhances the efficacy of chemotherapy in the autochthonous genetically engineered KPC model, while also demonstrating antifibrotic effects in human patient-derived xenograft models of pancreatic cancer. PXS-5505 is orally bioavailable, safe and effective at inhibiting lysyl oxidase activity in tissues. Our findings present the rationale for progression of a pan-lysyl oxidase inhibitor aimed at eliciting a reduction in stromal matrix to potentiate chemotherapy in pancreatic ductal adenocarcinoma., (© 2023. The Author(s).)

The tumour stroma, and in particular the extracellular matrix (ECM), is a salient feature of solid tumours that plays a crucial role in shaping their progression. Many desmoplastic tumours including breast cancer involve the significant accumulation of type I collagen. However, recently it has become clear that the precise distribution and organisation of matrix molecules such as collagen I is equally as important in the tumour as their abundance. Cancer-associated fibroblasts (CAFs) coexist within breast cancer tissues and play both pro- and anti-tumourigenic roles through remodelling the ECM. Here, using temporal proteomic profiling of decellularized tumours, we interrogate the evolving matrisome during breast cancer progression. We identify 4 key matrisomal clusters, and pinpoint collagen type XII as a critical component that regulates collagen type I organisation. Through combining our proteomics with single-cell transcriptomics, and genetic manipulation models, we show how CAF-secreted collagen XII alters collagen I organisation to create a pro-invasive microenvironment supporting metastatic dissemination. Finally, we show in patient cohorts that collagen XII may represent an indicator of breast cancer patients at high risk of metastatic relapse., (© 2022. The Author(s).)

Cancer-associated fibroblasts (CAF) are major contributors to pancreatic ductal adenocarcinoma (PDAC) progression through protumor signaling and the generation of fibrosis, the latter of which creates a physical barrier to drugs. CAF inhibition is thus an ideal component of any therapeutic approach for PDAC. SLC7A11 is a cystine transporter that has been identified as a potential therapeutic target in PDAC cells. However, no prior study has evaluated the role of SLC7A11 in PDAC tumor stroma and its prognostic significance. Here we show that high expression of SLC7A11 in human PDAC tumor stroma, but not tumor cells, is independently prognostic of poorer overall survival. Orthogonal approaches showed that PDAC-derived CAFs are highly dependent on SLC7A11 for cystine uptake and glutathione synthesis and that SLC7A11 inhibition significantly decreases CAF proliferation, reduces their resistance to oxidative stress, and inhibits their ability to remodel collagen and support PDAC cell growth. Importantly, specific ablation of SLC7A11 from the tumor compartment of transgenic mouse PDAC tumors did not affect tumor growth, suggesting the stroma can substantially influence PDAC tumor response to SLC7A11 inhibition. In a mouse orthotopic PDAC model utilizing human PDAC cells and CAFs, stable knockdown of SLC7A11 was required in both cell types to reduce tumor growth, metastatic spread, and intratumoral fibrosis, demonstrating the importance of targeting SLC7A11 in both compartments. Finally, treatment with a nanoparticle gene-silencing drug against SLC7A11, developed by our laboratory, reduced PDAC tumor growth, incidence of metastases, CAF activation, and fibrosis in orthotopic PDAC tumors. Overall, these findings identify an important role of SLC7A11 in PDAC-derived CAFs in supporting tumor growth. SIGNIFICANCE: This study demonstrates that SLC7A11 in PDAC stromal cells is important for the tumor-promoting activity of CAFs and validates a clinically translatable nanomedicine for therapeutic SLC7A11 inhibition in PDAC., (©2021 American Association for Cancer Research.)

The lysyl oxidase (LOX) family of enzymes are a major driver in the biogenesis of desmoplastic matrix at the primary tumour and secondary metastatic sites. With the increasing interest in and development of anti-stromal therapies aimed at improving clinical outcomes of cancer patients, the Lox family has emerged as a potentially powerful clinical target. This review examines how lysyl oxidase family dysregulation in solid cancers contributes to disease progression and poor patient outcomes, as well as an evaluation of the preclinical landscape of LOX family targeting therapeutics. We also discuss the suitability of the LOX family as a diagnostic and/or prognostic marker in solid tumours.

Background: The use of in vitro cell cultures is a powerful tool for obtaining key insights into the behaviour and response of cells to interventions in normal and disease situations. Unlike in vivo settings, in vitro experiments allow a fine-tuned control of a range of microenvironmental elements independently within an isolated setting. The recent expansion in the use of three-dimensional (3D) in vitro assays has created a number of representative tools to study cell behaviour in a more physiologically 3D relevant microenvironment. Complex 3D in vitro models that can recapitulate human tissue biology are essential for understanding the pathophysiology of disease., Aim: The development of the 3D coculture collagen contraction and invasion assay, the "organotypic assay," has been widely adopted as a powerful approach to bridge the gap between standard two-dimensional tissue culture and in vivo mouse models. In the cancer setting, these assays can then be used to dissect how stromal cells, such as cancer-associated fibroblasts (CAFs), drive extracellular matrix (ECM) remodelling to alter cancer cell behaviour and response to intervention. However, to date, many of the published organotypic protocols are low-throughput, time-consuming (up to several weeks), and work-intensive with often limited scalability. Our aim was to develop a fast, high-throughput, scalable 3D organotypic assay for use in oncology screening and drug development., Methods and Results: Here, we describe a modified 96-well organotypic assay, the "Mini-Organo," which can be easily completed within 5 days. We demonstrate its application in a wide range of mouse and human cancer biology approaches including evaluation of stromal cell 3D ECM remodelling, 3D cancer cell invasion, and the assessment of efficacy of potential anticancer therapeutic targets. Furthermore, the organotypic assay described is highly amenable to customisation using different cell types under diverse experimental conditions., Conclusions: The Mini-Organo high-throughput 3D organotypic assay allows the rapid screening of potential cancer therapeutics in human and mouse models in a time-efficient manner., (© 2019 The Authors Cancer Reports Published by Wiley Periodicals, Inc.)

The extracellular matrix (ECM) is a fundamental component of tissue microenvironments and its dysregulation has been implicated in a number of diseases, in particular cancer. Tumour desmoplasia (fibrosis) accompanies the progression of many solid cancers, and is also often induced as a result of many frontline chemotherapies. This has recently led to an increased interest in targeting the underlying processes. The major structural components of the ECM contributing to desmoplasia are the fibrillar collagens, whose key assembly mechanism is the enzymatic stabilisation of procollagen monomers by the lysyl oxidases. The lysyl oxidase family of copper-dependent amine oxidase enzymes are required for covalent cross-linking of collagen (as well as elastin) molecules into the mature ECM. This key step in the assembly of collagens is of particular interest in the cancer field since it is essential to the tumour desmoplastic response. LOX family members are dysregulated in many cancers and consequently the development of small molecule inhibitors targeting their enzymatic activity has been initiated by many groups. Development of specific small molecule inhibitors however has been hindered by the lack of crystal structures of the active sites, and therefore alternate indirect approaches to target LOX have also been explored. In this review, we introduce the importance of, and assembly steps of the ECM in the tumour desmoplastic response focussing on the role of the lysyl oxidases. We also discuss recent progress in targeting this family of enzymes as a potential therapeutic approach., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Inhibition of the NLRP3 inflammasome is a promising strategy for the development of new treatments for inflammatory diseases. MCC950 is a potent and specific small-molecule inhibitor of the NLRP3 pathway, but its molecular target is not defined. Here, we show that MCC950 directly interacts with the Walker B motif within the NLRP3 NACHT domain, thereby blocking ATP hydrolysis and inhibiting NLRP3 activation and inflammasome formation.

The fertilizing properties of bird manure, or guano, have played an important role in plant cultivation for thousands of years. Research into its chemical composition by Unger in 1846 identified a novel compound, now known as guanine, a purine base that is essential for DNA and RNA biosynthesis and cell signalling. Nitrogen-rich guano can also harbour human pathogens, one significant example being the fungal pathogen Cryptococcus neoformans. Historically associated with pigeon droppings, C. neoformans is able to infect immunocompromised individuals with the aid of a number of adaptive virulence traits. To gain insight into this niche, a quantitative analysis of pigeon guano was performed by LC/MS to determine the concentrations of purines present. Guanine was found in abundance, in particular, in aged guano samples that contained 156-296 μg/g [w/w] compared to 75 μg/g in fresh guano. Adenine concentrations were more consistent between fresh and aged samples, 13 μg/g compared to 10-15 μg/g, respectively. C. neoformans strains that lack key enzymes of the de novo purine synthesis pathway and are guanine or adenine auxotrophs displayed differences in their ability to exploit this substrate: growth of a guanine auxotrophic mutant (gua1Δ) was partially restored on 30% pigeon guano media, but an adenine auxotrophic mutant (ade13Δ) was unable to grow. We conclude that while purine salvage is likely a useful resource-saving mechanism, alone it is not sufficient to fully provide the purines required by wild-type C. neoformans growing in its guano niche.

Tumour metastasis is a dynamic and systemic process. It is no longer seen as a tumour cell-autonomous program but as a multifaceted and complex series of events, which is influenced by the intrinsic cellular mutational burden of cancer cells and the numerous bidirectional interactions between malignant and non-malignant cells and fine-tuned by the various extrinsic cues of the extracellular matrix. In cancer biology, metastasis as a process is one of the most technically challenging aspects of cancer biology to study. As a result, new platforms and technologies are continually being developed to better understand this process. In this review, we discuss some of the recent advances in metastasis and how the information gleaned is re-shaping our understanding of metastatic dissemination., Competing Interests: No competing interests were disclosed.

The extracellular matrix (ECM) is present in all solid tissues and considered a master regulator of cell behaviour and phenotype. The importance of maintaining the correct biochemical and biophysical properties of the ECM, and the subsequent regulation of cell and tissue homeostasis, is illustrated by the simple fact that the ECM is highly dysregulated in many different types of disease, especially cancer. The loss of tissue ECM homeostasis and integrity is seen as one of the hallmarks of cancer and typically defines transitional events in progression and metastasis. The vast majority of cancer studies place an emphasis on exploring the behaviour and intrinsic signalling pathways of tumour cells. Their goal was to identify ways to target intracellular pathways regulating cancer. Cancer progression and metastasis are powerfully influenced by the ECM and thus present a vast, unexplored repository of anticancer targets that we are only just beginning to tap into. Deconstructing the complexity of the tumour ECM landscape and identifying the interactions between the many cell types, soluble factors and extracellular-matrix proteins have proved challenging. Here, we discuss some of the emerging tools and platforms being used to catalogue and chart the ECM in cancer., (© 2018 The Authors. International Journal of Experimental Pathology © 2018 International Journal of Experimental Pathology.)

Resistance to antimicrobials is a growing problem in both developed and developing countries. In nations where AIDS is most prevalent, the human fungal pathogen Cryptococcus neoformans is a significant contributor to mortality, and its growing resistance to current antifungals is an ever-expanding threat. We investigated octapeptin C4, from the cationic cyclic lipopeptide class of antimicrobials, as a potential new antifungal. Octapeptin C4 was a potent, selective inhibitor of this fungal pathogen with an MIC of 1.56 μg/ml. Further testing of octapeptin C4 against 40 clinical isolates of C. neoformans var. grubii or neoformans showed an MIC of 1.56 to 3.13 μg/ml, while 20 clinical isolates of C. neoformans var. gattii had an MIC of 0.78 to 12.5 μg/ml. In each case, the MIC values for octapeptin C4 were equivalent to, or better than, current antifungal drugs fluconazole and amphotericin B. The negatively charged polysaccharide capsule of C. neoformans influences the pathogen's sensitivity to octapeptin C4, whereas the degree of melanization had little effect. Testing synthetic octapeptin C4 derivatives provided insight into the structure activity relationships, revealing that the lipophilic amino acid moieties are more important to the activity than the cationic diaminobutyric acid groups. Octapeptins have promising potential for development as anticryptococcal therapeutic agents., (Copyright © 2018 Chitty et al.)

There is significant clinical need for new antifungal agents to manage infections with pathogenic species such as Cryptococcus neoformans Because the purine biosynthesis pathway is essential for many metabolic processes, such as synthesis of DNA and RNA and energy generation, it may represent a potential target for developing new antifungals. Within this pathway, the bifunctional enzyme adenylosuccinate (ADS) lyase plays a role in the formation of the key intermediates inosine monophosphate and AMP involved in the synthesis of ATP and GTP, prompting us to investigate ADS lyase in C. neoformans. Here, we report that ADE13 encodes ADS lyase in C. neoformans. We found that an ade13 Δ mutant is an adenine auxotroph and is unable to successfully cause infections in a murine model of virulence. Plate assays revealed that production of a number of virulence factors essential for dissemination and survival of C. neoformans in a host environment was compromised even with the addition of exogenous adenine. Purified recombinant C. neoformans ADS lyase shows catalytic activity similar to its human counterpart, and its crystal structure, the first fungal ADS lyase structure determined, shows a high degree of structural similarity to that of human ADS lyase. Two potentially important amino acid differences are identified in the C. neoformans crystal structure, in particular a threonine residue that may serve as an additional point of binding for a fungal enzyme-specific inhibitor. Besides serving as an antimicrobial target, C. neoformans ADS lyase inhibitors may also serve as potential therapeutics for metabolic disease; rather than disrupt ADS lyase, compounds that improve the stability the enzyme may be used to treat ADS lyase deficiency disease., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

While members of the Kingdom Fungi are found across many of the world's most hostile environments, only a limited number of species can thrive within the human host. The causative agents of the most common invasive fungal infections are Candida albicans , Aspergillus fumigatus , and Cryptococcus neoformans . During the infection process, these fungi must not only combat the host immune system while adapting to dramatic changes in temperature and pH, but also acquire sufficient nutrients to enable growth and dissemination in the host. One class of nutrients required by fungi, which is found in varying concentrations in their environmental niches and the human host, is the purines. These nitrogen-containing heterocycles are one of the most abundant organic molecules in nature and are required for roles as diverse as signal transduction, energy metabolism and DNA synthesis. The most common life-threatening fungal pathogens can degrade, salvage and synthesize de novo purines through a number of enzymatic steps that are conserved. While these enable them to adapt to the changing purine availability in the environment, only de novo purine biosynthesis is essential during infection and therefore an attractive antimycotic target., Competing Interests: The authors declare no conflict of interest.

Virulence of Cryptococcus neoformans is regulated by a range of transcription factors, and is also influenced by the acquisition of adaptive mutations during infection. Beyond the temporal regulation of virulence factor production by transcription factors and these permanent microevolutionary changes, heritable epigenetic modifications such as histone deacetylation may also play a role during infection. Here we describe the first comprehensive analysis of the sirtuin class of NAD+ dependent histone deacetylases in the phylum Basidiomycota, identifying five sirtuins encoded in the C. neoformans genome. Each sirtuin gene was deleted and a wide range of phenotypic tests performed to gain insight into the potential roles they play. Given the pleiotropic nature of sirtuins in other species, it was surprising that only two of the five deletion strains revealed mutant phenotypes in vitro. However, cryptic consequences of the loss of each sirtuin were identified through whole cell proteomics, and mouse infections revealed a role in virulence for SIR2, HST3 and HST4. The most intriguing phenotype was the repeated inability to complement mutant phenotypes through the reintroduction of the wild-type gene. These data support the model that regulation of sirtuin activity may be employed to enable a drastic alteration of the epigenetic landscape and virulence of C. neoformans.

Over the last four decades the HIV pandemic and advances in medical treatments that also cause immunosuppression have produced an ever-growing cohort of individuals susceptible to opportunistic pathogens. Of these, AIDS patients are particularly vulnerable to infection by the encapsulated yeast Cryptococcus neoformans Most commonly found in the environment in purine-rich bird guano, C. neoformans experiences a drastic change in nutrient availability during host infection, ultimately disseminating to colonize the purine-poor central nervous system. Investigating the consequences of this challenge, we have characterized C. neoformans GMP synthase, the second enzyme in the guanylate branch of de novo purine biosynthesis. We show that in the absence of GMP synthase, C. neoformans becomes a guanine auxotroph, the production of key virulence factors is compromised, and the ability to infect nematodes and mice is abolished. Activity assays performed using recombinant protein unveiled differences in substrate binding between the C. neoformans and human enzymes, with structural insights into these kinetic differences acquired via homology modeling. Collectively, these data highlight the potential of GMP synthase to be exploited in the development of new therapeutic agents for the treatment of disseminated, life-threatening fungal infections., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Opportunistic fungal pathogens such as Cryptococcus neoformans are a growing cause of morbidity and mortality among immunocompromised populations worldwide. To address the current paucity of antifungal therapeutic agents, further research into fungal-specific drug targets is required. Adenylosuccinate synthetase (AdSS) is a crucial enzyme in the adeosine triphosphate (ATP) biosynthetic pathway, catalyzing the formation of adenylosuccinate from inosine monophosphate and aspartate. We have investigated the potential of this enzyme as an antifungal drug target, finding that loss of function results in adenine auxotrophy in C. neoformans, as well as complete loss of virulence in a murine model. Cryptococcal AdSS was expressed and purified in Escherichia coli and the enzyme's crystal structure determined, the first example of a structure of this enzyme from fungi. Together with enzyme kinetic studies, this structural information enabled comparison of the fungal enzyme with the human orthologue and revealed species-specific differences potentially exploitable via rational drug design. These results validate AdSS as a promising antifungal drug target and lay a foundation for future in silico and in vitro screens for novel antifungal compounds.

Canine obesity is increasing in prevalence in the UK and raises concerns about dog welfare. This study compares the health-related quality of life (HRQL) of dogs of varying body condition: overweight and obese (BCS 4 and 5) versus non-overweight dogs (BCS 2 and 3), obese (BCS 5) versus non-overweight (BCS 2 and 3) and an overall comparison between all four BCS (BCS 2, 3, 4 and 5) using a novel, validated HRQL instrument which is both web and mobile tablet/phone app based. Of 271 dog owners who were approached, 174 completed a web-based instrument (2013) or a mobile tablet app instrument (2014) during the summers of 2013 and 2014. Automatically generated scores in four domains of HRQL (energetic/enthusiastic, happy/content, active/comfortable, calm/relaxed) were compared for dogs with each of the body condition scores (BCS 2-5). For all body condition scores a statistically significant difference was found between the HRQL scores in two of the domains: energetic/enthusiastic (p=0.02) and active comfortable (p=0.004). When BCS 2 and 3 were compared to BCS 4 and 5, statistical significance was found in the same two domains - energetic/enthusiastic (p=0.01) and active comfortable (p=0.001) - as it was in comparison of non-overweight (BCS 2 and 3) compared to obese dogs (BCS 5): energetic/enthusiastic (p=0.012) and active comfortable (p=0.004). These results suggest that overweight and obese dogs have a reduced HRQL in two of the domains compared to non-overweight dogs, and that differences in HRQL are detectable between BCS scores 2, 3, 4 and 5., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Eighteen natural products sourced from Australian micro- or macro-fungi were screened for antibacterial and antifungal activity. This focused library was comprised of caprolactams, polyamines, quinones, and polyketides, with additional large-scale isolation studies undertaken in order to resupply previously identified compounds. Chemical investigations of the re-fermented culture from the endophytic fungus Pestalotiopsis sp. yielded three caprolactam analogues, pestalactams D-F, along with larger quantities of the known metabolite pestalactam A, which was methylated using diazomethane to yield 4-O-methylpestalactam A. The chemical structures of the previously undescribed fungal metabolites were determined by analysis of 1D/2D NMR and MS data. The structure of 4-O-methylpestalactam A was confirmed following single crystal X-ray diffraction analysis. The antibacterial and antifungal activity of all compounds was assessed, which identified three compounds, (1S,3R)-austrocortirubin, (1S,3S)-austrocortirubin, and 1-deoxyaustrocortirubin with mild activity (100 μM) against Gram-positive isolates and one compound, 2-hydroxy-6-methyl-8-methoxy-9-oxo-9H-xanthene-1-carboxylic acid, with activity against Cryptococcus neoformans and Cryptococcus gattii at 50 μM., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Just as Koch's postulates formed the foundation of early infectious disease study, Stanley Falkow's molecular Koch's postulates define best practice in determining whether a specific gene contributes to virulence of a pathogen. Fundamentally, these molecular postulates state that if a gene is involved in virulence, its removal will compromise virulence. Likewise, its reintroduction should restore virulence to the mutant. These approaches are widely employed in Cryptococcus neoformans, where gene deletion via biolistic transformation is a well-established technique. However, the complementation of these mutants is less straightforward. Currently, one of three approaches will be taken: the gene is reintroduced at the original locus, the gene is reintroduced into a random site in the genome, or the mutant is not complemented at all. Depending on which approach is utilized, the mutant may be complemented but other genes are potentially disrupted in the process. To counter the drawbacks of the current approaches to complementation we have created a new tool to assist in this key step in the study of a gene's role in virulence. We have identified and characterized a small gene-free region in the C. neoformans genome dubbed the "safe haven", and constructed a plasmid vector that targets DNA constructs to this preselected site. The plasmid vector integrates with high frequency, effectively complementing a mutant strain without disrupting adjacent genes. qRT-PCR of the flanking genes on either side of the safe haven site following integration of the targeting vector revealed no changes in their expression, and no secondary phenotypes were observed in a range of phenotypic assays including an intranasal murine infection model. Combined, these data confirm that we have successfully created a much-needed molecular resource for the Cryptococcus community, enabling the reliable fulfillment of the molecular Koch's postulates.