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Supplemental Methods, Supplemental References, Supplemental Figure Legends, Figure S1-13, Table S1-5 and S14

cfDNA variants associated with resistance to on-label targeted therapies across six cancer types

Driver alteration prevalence in the cfDNA cohort versus in TCGA

Purpose: Cell-free DNA (cfDNA) sequencing provides a noninvasive method for obtaining actionable genomic information to guide personalized cancer treatment, but the presence of multiple alterations in circulation related to treatment and tumor heterogeneity complicate the interpretation of the observed variants.Experimental Design: We describe the somatic mutation landscape of 70 cancer genes from cfDNA deep-sequencing analysis of 21,807 patients with treated, late-stage cancers across >50 cancer types. To facilitate interpretation of the genomic complexity of circulating tumor DNA in advanced, treated cancer patients, we developed methods to identify cfDNA copy-number driver alterations and cfDNA clonality.Results: Patterns and prevalence of cfDNA alterations in major driver genes for non–small cell lung, breast, and colorectal cancer largely recapitulated those from tumor tissue sequencing compendia (The Cancer Genome Atlas and COSMIC; r = 0.90–0.99), with the principal differences in alteration prevalence being due to patient treatment. This highly sensitive cfDNA sequencing assay revealed numerous subclonal tumor-derived alterations, expected as a result of clonal evolution, but leading to an apparent departure from mutual exclusivity in treatment-naïve tumors. Upon applying novel cfDNA clonality and copy-number driver identification methods, robust mutual exclusivity was observed among predicted truncal driver cfDNA alterations (FDR = 5 × 10−7 for EGFR and ERBB2), in effect distinguishing tumor-initiating alterations from secondary alterations. Treatment-associated resistance, including both novel alterations and parallel evolution, was common in the cfDNA cohort and was enriched in patients with targetable driver alterations (>18.6% patients).Conclusions: Together, these retrospective analyses of a large cfDNA sequencing data set reveal subclonal structures and emerging resistance in advanced solid tumors. Clin Cancer Res; 24(15); 3528–38. ©2018 AACR.

Mutual exclusivity statistics for cfDNA alterations in lung adenocarcinoma, breast cancer, and colorectal cancer

Background Gallium is a semi-metallic element known since the 1930s to have antimicrobial activity. This activity stems primarily from gallium's ability to mimic trivalent iron and disrupt specific Fe(III)-dependent pathways, particularly DNA synthesis (due to inhibition of ribonucleotide reductase). Because of its novel mechanism of action, gallium is currently being investigated as a new antibacterial agent, particularly in light of the increasing resistance of many pathogenic bacteria to existing antibiotics. Gallium maltolate (GaM) is being developed as an orally and topically administrable form of gallium. Yaws is a neglected tropical disease affecting mainly the skin and skeletal system of children in underprivileged settings. It is currently the object of a WHO-promoted eradication campaign using mass administration of the macrolide azithromycin, an antibiotic to which the yaws agent Treponema pallidum subsp. pertenue has slowly begun to develop genetic resistance. Methods Because yaws transmission is mainly due to direct skin contact with an infectious skin lesion, we evaluated the treponemicidal activity of GaM applied topically to skin lesions in a rabbit model of yaws. Treatment efficacy was evaluated by measuring lesion diameter, treponemal burden in lesion aspirates as determined by dark field microscopy and amplification of treponemal RNA, serology, and immunohistochemistry of biopsied tissue samples. Results Our results show that topical GaM was effective in reducing treponemal burden in yaws experimental lesions, particularly when applied at the first sign of lesion appearance but, as expected, did not prevent pathogen dissemination. Conclusion Early administration of GaM to yaws lesions could reduce the infectivity of the lesions and thus yaws transmission, potentially contributing to current and future yaws control campaigns., Author summary Yaws is a neglected tropical disease affecting children in underprivileged countries, transmitted through direct skin contact with an active lesion. This infection, although rarely fatal, can lead to disfigurement and serious disability. The World Health Organization is currently conducting a yaws eradication effort that employs mass administration of azithromycin, an antibiotic against which the yaws pathogen has slowly begun to develop genetic resistance. Because this phenomenon has the potential to undermine the eradication effort, we investigated the antimicrobial activity of gallium maltolate, which has a novel mechanism of action, against the yaws pathogen. Our initial results show that topical application of gallium maltolate has significant treponemicidal activity, and suggest that this compound might find an application in the effort to eradicate yaws. Future studies will evaluate whether oral administration of gallium maltolate is as effective as the antibiotics currently approved for yaws treatment to clear systemic infection.

Background Low-density lipoprotein cholesterol (LDL-C) lowering is the primary objective of patient management for cardiovascular disease. However, large numbers of patients who have achieved their LDL-C goal remain at risk for cardiovascular events. Low-density lipoprotein subfractions may provide insight into this residual risk. Thus, LDL subfraction standardization and consistency are critical to these efforts. Aim This study aimed to determine the agreement of the analytical results among 4 methods commonly used for LDL subfractionation, namely, segmented gradient gel electrophoresis (sGGE), ultracentrifugation-vertical auto profile (VAP), nuclear magnetic resonance (NMR), and ion mobility (IM). Methods Blood samples were collected from 228 apparently healthy adults and sent to 4 clinical reference laboratories for analysis. The LDL phenotype was reported as pattern A (larger, less dense particles) or pattern B (smaller, more dense particles), respectively, and was the primary measure of comparison. An intermediate pattern (A/B) was also reported for sGGE and VAP. Results We observed complete agreement in the LDL phenotype among the 4 methods in 64% of subjects and agreement among at least 3 of the 4 methods in 87% of subjects. Agreement among pairs of methods ranged from 73% to 98% depending on how differences in reporting of subjects with intermediate results were considered. When subjects having intermediate A/B pattern were excluded, sGGE and IM had the highest agreement (98%) of any pair of methods. Conclusions We found substantial agreement in the reported LDL phenotype among 4 LDL subfraction measurement technologies as performed by different clinical reference laboratories.

Patients with nonalcoholic fatty liver disease (NAFLD) are at increased risk of cardiovascular disease, and atherogenic lipoproteins may play an important role.The objective of the study was to determine the contribution of the severity of steatohepatitis to atherogenic dyslipidemia in patients with NAFLD.This was a cross-sectional study.The study was conducted at a university hospital.Patients were recruited from outpatient clinics or from the general population (n = 188).Measurement of hepatic triglyceride content by magnetic resonance spectroscopy, histology (liver biopsy), metabolic profile by means of an oral glucose tolerance test, and lipoprotein analyses were performed.Outcomes measured included standard lipids, lipoprotein subfraction analysis (apolipoprotein B/A1 levels, low-density lipoprotein (LDL) particle size/phenotype, and LDL/high-density lipoprotein subfractions), and insulin resistance.Patients with NAFLD had severe insulin resistance, especially at the level of the adipose tissue, when compared with patients without NAFLD. Despite small differences in triglycerides and high-density lipoprotein-cholesterol, patients with NAFLD had a significantly higher plasma apolipoprotein B to apolipoprotein A1 ratio (0.66 ± 0.02 vs 0.58 ± 0.02, P = .01) and smaller LDL particle size (216.2 ± 0.7 vs 219.4 ± 1.1 Å, P = .01). Of note, these differences between patients with/without NAFLD were independent of the presence of obesity. Severity of steatohepatitis did not significantly influence the lipoprotein profile. Worse atherogenic dyslipidemia was best predicted by the degree of liver fat accumulation and adipose tissue and systemic insulin resistance.NAFLD was associated with a worse atherogenic lipoprotein profile, regardless of similar body mass index and other clinical parameters. We speculate that this lipoprotein profile is driven mostly by liver fat content and insulin resistance and appears not to be worsened by obesity or the severity of liver disease (nonalcoholic steatohepatitis).

Background: VLDL and chylomicrons may interfere with measurements of apolipoprotein B (apo B) on LDL particles. Ultracentrifugation of samples enriched in chylomicrons and VLDL and subsequent measurement of apo B in the infranate fraction [density (d) = 1.006] removes this interference. This apo B fraction is called “LDL–apo B.” Methods: We retrospectively analyzed 64 895 measurements of triglycerides, total apo B, and LDL–apo B. Samples were ultracentrifuged, and 3 commercially available immunoassays that use different antibodies were used to measure LDL–apo B in the 1.006 infranate fraction. Results: After adjusting for triglyceride concentration, we found total apo B and LDL–apo B measurements to be strongly correlated. We derived a simple linear equation for calculating LDL–apo B concentration (in milligrams per deciliter) from measurements of total apo B and triglycerides: LDL–apo B = apo B − 10 mg/dL − triglycerides/32. This equation accurately predicts LDL–apo B values within ±12% of the measured value in 75% of cases. Conclusions: Our equation provides a convenient means of estimating LDL–apo B from commonly available measurements of total apo B and triglycerides without the need for ultracentrifugation. LDL–apo B measurements were also independent of the different apo B antibodies in the 3 assays used in this study. An equation that predicts LDL–apo B particle number may be useful, regardless of the apo B assay used.

The enzyme 7,8-dihydropteroate synthase (DHPS) catalyzes the condensation of para-aminobenzoic acid (pABA) with 6-hydroxymethyl-7, 8-dihydropterin-pyrophosphate to form 7,8-dihydropteroate and pyrophosphate. DHPS is essential for the de novo synthesis of folate in prokaryotes, lower eukaryotes, and in plants, but is absent in mammals. Inhibition of this enzyme's activity by sulfonamide and sulfone drugs depletes the folate pool, resulting in growth inhibition and cell death. Here, we report the 1.7 A resolution crystal structure of the binary complex of 6-hydroxymethylpterin monophosphate (PtP) with DHPS from Mycobacterium tuberculosis (Mtb), a pathogen responsible for the death of millions of human beings each year. Comparison to other DHPS structures reveals that the M. tuberculosis DHPS structure is in a unique conformation in which loop 1 closes over the active site. The Mtb DHPS structure hints at a mechanism in which both loops 1 and 2 play important roles in catalysis by shielding the active site from bulk solvent and allowing pyrophosphoryl transfer to occur. A binding mode for pABA, sulfonamides and sulfones is suggested based on: (i) the new conformation of the closed loop 1; (ii) the distribution of dapsone and sulfonamide resistance mutations; (iii) the observed direction of the bond between the 6-methyl carbon atom and the bridging oxygen atom to the alpha-phosphate group in the Mtb DHPS:PtP binary complex; and (iv) the conformation of loop 2 in the Escherichia coli DHPS structure. Finally, the Mtb DHPS structure reveals a highly conserved pterin binding pocket that may be exploited for the design of novel antimycobacterial agents.

Parasite genes often use codons which are rarely used in the highly expressed genes of Escherichia coli, possibly resulting in translational stalling and lower yields of recombinant protein. We have constructed the "RIG" plasmid to overcome the potential codon-bias problem seen in Plasmodium genes. RIG contains the genes that encode three tRNAs (Arg, Ile, Gly), which recognise rare codons found in parasite genes. When co-transformed into E. coli along with expression plasmids containing parasite genes, RIG can greatly increase levels of overexpressed protein. Codon frequency analysis suggests that RIG may be applied to a variety of protozoan and helminth genes.

A recently developed spectroscopic technique was used to determine orygen diffusion coefficients, D, as a function of temperature in polystyrene and polycarbonate films. The data were quantified by using an Arrhenius expression with the following variables: (a) a diffusion activation barrier, E act , and (b) a diffusion coefficient, D 0 , that represents the condition of «barrier-free» gas transport. From the perspective that diffusion depends on free volume, the parameters E act and D 0 are interpreted to reflect dynamic and static elements of free volume in the polymer matrix. The addition of low molecular weight solutes to these amorphous glasses can alter the oxygen diffusion coefficient by affecting the dynamic and/or static free volume of the material

Chemotherapy against human African trypanosomiasis relies on four drugs that cause frequent and occasionally severe side-effects. Because human African trypanosomiasis is a disease of poor people in Africa, the traditional market-driven pathways to drug development are not available. One potentially rapid and cost-effective approach to identifying and developing new trypanocidal drugs would be high throughput-screening of existing drugs already approved for other uses, as well as clinical candidates in late development. We have developed an ATP-bioluminescence assay that could be used to rapidly and efficiently screen compound libraries against trypanosomes in a high throughput-screening format to validate this notion. We screened a collection of 2160 FDA-approved drugs, bioactive compounds and natural products to identify hits that were cytotoxic to cultured Trypanosoma brucei at a concentration of 1 mum or less. This meant that any hit identified would be effective at a concentration readily achievable by standard drug dosing in humans. From the screen, 35 hits from seven different drug categories were identified. These included the two approved trypanocidal drugs, suramin and pentamidine, several other drugs suspected but never validated as trypanocidal, and 17 novel trypanocidal drugs.

We report here the failure of the Synchron LX (Beckman) and Vitros 950 (Johnson & Johnson) assays to detect HDL-cholesterol (HDL-C) in the presence of a paraprotein. HDL-C was repeatedly undetectable (