Human and animal fungal pathogens are a growing threat worldwide leading to emerging infections and creating new risks for established ones. There is a growing need for a rapid and accurate identification of pathogens to enable early diagnosis and targeted antifungal therapy. Morphological and biochemical identification methods are time-consuming and require trained experts. Alternatively, molecular methods, such as DNA barcoding, a powerful and easy tool for rapid monophasic identification, offer a practical approach for species identification and less demanding in terms of taxonomical expertise. However, its wide-spread use is still limited by a lack of quality-controlled reference databases and the evolving recognition and definition of new fungal species/complexes. An international consortium of medical mycology laboratories was formed aiming to establish a quality controlled ITS database under the umbrella of the ISHAM working group on "DNA barcoding of human and animal pathogenic fungi." A new database, containing 2800 ITS sequences representing 421 fungal species, providing the medical community with a freely accessible tool at http://www.isham.org/ and http://its.mycologylab.org/ to rapidly and reliably identify most agents of mycoses, was established. The generated sequences included in the new database were used to evaluate the variation and overall utility of the ITS region for the identification of pathogenic fungi at intra-and interspecies level. The average intraspecies variation ranged from 0 to 2.25%. This highlighted selected pathogenic fungal species, such as the dermatophytes and emerging yeast, for which additional molecular methods/genetic markers are required for their reliable identification from clinical and veterinary specimens.

Cryptococcosis is an important fungal infection in immunocompromised individuals, especially those infected with HIV. In Brazil, despite the free availability of antiretroviral therapy (ART) in the public health system, the mortality rate due to Cryptococcus neoformans meningitis is still high. To obtain a more detailed picture of the population genetic structure of this species in southeast Brazil, we studied 108 clinical isolates from 101 patients and 35 environmental isolates. Among the patients, 59% had a fatal outcome mainly in HIV-positive male patients. All the isolates were found to be C. neoformans var. grubii major molecular type VNI and mating type locus alpha. Twelve were identified as diploid by flow cytometry, being homozygous (AαAα) for the mating type and by PCR screening of the STE20, GPA1, and PAK1 genes. Using the ISHAM consensus multilocus sequence typing (MLST) scheme, 13 sequence types (ST) were identified, with one being newly described. ST93 was identified from 81 (75%) of the clinical isolates, while ST77 and ST93 were identified from 19 (54%) and 10 (29%) environmental isolates, respectively. The southeastern Brazilian isolates had an overwhelming clonal population structure. When compared with populations from different continents based on data extracted from the ISHAM-MLST database (mlst.mycologylab.org) they showed less genetic variability. Two main clusters within C. neoformans var. grubii VNI were identified that diverged from VNB around 0.58 to 4.8 million years ago.

Galleria mellonella larvae have been widely used as alternative non-mammalian models for the study of fungal virulence and pathogenesis. The larvae can be acquired in small volumes from worm farms, pet stores, or other independent suppliers commonly found in the United States and parts of Europe. However, in countries with no or limited commercial availability, the process of shipping these larvae can cause them stress, resulting in decreased or altered immunity. Furthermore, the conditions used to rear these larvae including diet, humidity, temperature, and maintenance procedures vary among the suppliers. Variation in these factors can affect the response of G. mellonella larvae to infection, thereby decreasing the reproducibility of fungal virulence experiments. There is a critical need for standardized procedures and incubation conditions for rearing G. mellonella to produce quality, unstressed larvae with the least genetic variability. In order to standardize these procedures, cost-effective protocols for the propagation and maintenance of G. mellonella larvae using an artificial diet, which has been successfully used in our own laboratory, requiring minimal equipment and expertise, are herein described. Examples for the application of this model in fungal pathogenicity and gene knockout studies as feasible alternatives for traditionally used animal models are also provided.

Evofosfamide (TH-302) is a hypoxia-activated DNA-crosslinking prodrug currently in clinical development for cancer therapy. Oxygen-sensitive activation of evofosfamide depends on oneelectron reduction, yet the reductases that catalyze this process in tumors are unknown. We used RNA sequencing, wholegenome CRISPR knockout, and reductase-focused short hairpin RNA screens to interrogate modifiers of evofosfamide activation in cancer cell lines. Involvement of mitochondrial electron transport in the activation of evofosfamide and the related nitroaromatic compounds EF5 and FSL-61 was investigated using 143B r 0 (r zero) cells devoid of mitochondrial DNA and biochemical assays in UT-SCC-74B cells. The potency of evofosfamide in 30 genetically diverse cancer cell lines correlated with the expression of genes involved in mitochondrial electron transfer. A whole-genome CRISPR screen in KBM-7 cells identified the DNA damage-response factors SLX4IP, C10orf90 (FATS), and SLFN11, in addition to the key regulator of mitochondrial function, YME1L1, and several complex I constituents as modifiers of evofosfamide sensitivity. A reductasefocused shRNA screen in UT-SCC-74B cells similarly identified mitochondrial respiratory chain factors. Surprisingly, 143B r 0 cells showed enhanced evofosfamide activation and sensitivity but had global transcriptional changes, including increased expression of nonmitochondrial flavoreductases. In UT-SCC-74B cells, evofosfamide oxidized cytochromes a, b, and c and inhibited respiration at complexes I, II, and IV without quenching reactive oxygen species production. Our results suggest that the mitochondrial electron transport chain contributes to evofosfamide activation and that predicting evofosfamide sensitivity in patients by measuring the expression of canonical bioreductive enzymes such as cytochrome P450 oxidoreductase is likely to be futile.

is to preserve Australian human and animal pathogenic fungal biodiversity while providing reference and clinical strains for the mycology community. The stored strains are identified phenotypically, biochemically and molecularly.They are stored either lyophilised, in glycerol at À808C or as living culture at 148C. The majority of the stored strains are the result of specific clinical, molecular epidemiological and basic science projects. As such, the pathogenic yeasts

Trastuzumab emtansine (T-DM1, Kadcyla) is an antibody-drug conjugate used in the treatment of HER2-positive breast cancer. However, its use is limited by acquired and intrinsic resistance, the mechanisms of which are not well understood. Further knowledge of T-DM1 resistance may provide new combination strategies or therapeutic targets to overcome resistance or new predictive biomarkers to identify the patients most likely to benefit from T-DM1 therapy. To discover genes responsible for T-DM1 sensitivity and resistance in an unbiased manner, we have conducted CRISPR/Cas9 functional genomics screens by a two-stage process. Firstly, we performed whole genome screens in MDA-MB-361 and MDA-MB-453 cells transduced with Cas9 and the GeCKOv2 lentiviral library that were exposed to T-DM1 and its effector DM1 for 8-13 weeks. Gene knockouts enriched or depleted in response to T-DM1 or DM1 treatment in either cell line were identified by sequencing of genomic DNA and differentially expressed genes by RNA sequencing, revealing 599 candidate genes of T-DM1 sensitivity. For high-throughput validation of the 599 genes, we developed a custom library of 2539 guide RNAs (gRNAs) to target these 599 genes, plus non-targeting controls. Cas9-expressing MDA-MB-361 cells were transduced with the custom library and exposed to T-DM1 for 28 days. MAGeCK analysis of gRNA sequencing revealed 11 genes that were significantly enriched and one gene that was significantly depleted at a false discovery rate (FDR) of <0.1. Two of the top hits in the secondary screen were two genes whose loss is known to promote T-DM1 resistance: ERBB2 (HER2) and SLC46A3 (P<8 × 10−5; FDR <0.007). Other top hits were TSC1 and TSC2 (P<3 × 10−6; FDR= 0.0004); which are both tumor suppressor genes and negative regulators of mTOR complex 1 (mTORC1). For subsequent validation, we have generated TSC2 knockout cell pools, which were more resistant to T-DM1 than wildtype cells in a competition growth assay. Knockout clones have been isolated and are being tested for T-DM1 resistance. Since mTOR inhibitors can phenocopy TSC1 and TSC2 by inhibiting mTORC1 activity, we have also evaluated T-DM1 in combination with the mTOR inhibitor KU-0063794 in a sulforhodamine B assay in MDA-MB-361 and MDA-MB-453 cells. Each agent potently inhibited cell proliferation and demonstrated synergistic anti-proliferative activity in combination. Together, our results suggest that TSC1 and TSC2 knockout may promote T-DM1 resistance and that targeting mTOR may be an effective strategy to overcome T-DM1 resistance. Citation Format: Francis W. Hunter, Barbara A. Lipert, Kyla N. Siemens, Aziza Khan, Hilary R. Barker, Troy W. Ketela, William R. Wilson, Tet-Woo Lee, Stephen M. Jamieson. Identification of TSC1 and TSC2 as potential determinants of sensitivity to trastuzumab emtansine [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5853.

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This abstract describes the rational development of novel drugs design for treating cancers based on the information provided by the sequencing of Human Genome. Out of 24,000 genes in our Genome, sixteen thousand genes code for good proteins that keep us healthy. Six thousand mutated genes are identified which are responsible for causing six thousand different diseases including cancers. The most obvious approach is to design drugs to shut off mutated genes that code for wrong protein. Professor Ross was the first person to use a highly toxic chemical called Nitrogen Mustard developed during the World Wars to shut off a cancer-causing gene. Nitrogen Mustard can cross-link double stranded DNA and shut off a gene. Unfortunately, it has no selectivity. It cross-links all rapidly dividing cells both normal and abnormal cells. This abstract also describes the use of a novel class of drugs called Aziridines which acts as a Prodrug. It does not attack all dividing cells, Aziridines are activated in the presence of acid. As cancer cells grow rapidly, they use Glucose as a source of energy. In cancer cells, Glucose is broken down to produce Lactic Acid. It is the acid that activate Aziridine ring which opens to attack a single strand of DNA shutting off the genes. Over the years, we developed over a hundred analogs of Aziridines and tested against an experimental animal tumor called the Walker Carcinoma 256 in Rats. One compound, Aziridine dinitro-benzamide (CB1854) is found to be seventy times more toxic to cancer cell. Using the same rationale, we translated animal work in human making AZQ (US Patent 4,146,622 & 4,233,215) for treating Glioblastoma, a solid aggressive brain tumor in human. A new approach to treat Breast Cancer is also described.