Global proteomic analysis of prenylated proteins in Plasmodium falciparum using an alkyne-modified isoprenoid analogue

Severe malaria due to Plasmodium falciparum infection remains a serious threat to health worldwide and new therapeutic targets are highly desirable. Small molecule inhibitors of prenyl transferases, enzymes that catalyze the post-translational isoprenyl modifications of proteins, exhibit potent antimalarial activity. The antimalarial actions of prenyltransferase inhibitors indicate that protein prenylation is required for malaria parasite development. In this study, we used a chemical biology strategy to experimentally characterize the entire complement of prenylated proteins in the human malaria parasite. In contrast to the expansive mammalian and fungal prenylomes, we find that P. falciparum possesses a restricted set of prenylated proteins. The prenylome of P. falciparum is dominated by Rab GTPases, in addition to a small number of prenylated proteins that also appear to function primarily in membrane trafficking. Overall, we found robust experimental evidence for a total of only thirteen prenylated proteins in P. falciparum, with suggestive evidence for an additional two probable prenyltransferase substrates. Our work contributes to an increasingly complete picture of essential, post-translational hydrophobic modifications in blood-stage P. falciparum

Post-operative ctDNA monitoring in stage I colon cancer: A case report

Circulating tumor DNA (ctDNA) level monitoring after surgery for colon cancer has been studied in stage II and III colon cancer to risk-stratify patients for adjuvant therapy. However, there is less data regarding the role of this diagnostic tool in the management of stage I disease, where current recommended surveillance is limited to screening colonoscopy at one year. In this report, we describe the case of a 57-year-old man with stage I colon cancer who underwent complete resection with adequate lymph node surgical sampling, normal preoperative CEA and no evidence of metastatic disease on initial imaging. The patient elected to undergo serial ctDNA monitoring after surgery. Rising ctDNA levels, five months after resection, prompted cross-sectional imaging which demonstrated metastatic disease to the liver. The patient subsequently received five cycles of leucovorin, 5-fluorouracil, oxaliplatin, and irinotecan with bevacizumab (FOLFOXIRI-Bev) and definitive microwave ablation to the liver metastases, with resulting undetectable ctDNA levels. The patient’s imaging and colonoscopy one-year post-operatively showed no evidence of disease, with ctDNA levels remaining undetectable. This report highlights the value of ctDNA monitoring in patients with early-stage colon cancer and suggests that further, large-scale studies may be warranted to determine its appropriate clinical use

ctDNA-based detection of molecular residual disease in stage I-III non-small cell lung cancer patients treated with definitive radiotherapy

BackgroundSensitive and reliable biomarkers for early detection of recurrence are needed to improve post-definitive radiation risk stratification, disease management, and outcomes for patients with unresectable early-stage or locally advanced non-small cell lung cancer (NSCLC) who are treated with definitive radiation therapy (RT). This prospective, multistate single-center, cohort study investigated the association of circulating tumor DNA (ctDNA) status with recurrence in patients with unresectable stage I-III NSCLC who underwent definitive RT.MethodsA total of 70 serial plasma samples from 17 NSCLC patients were collected before, during, and after treatment. A personalized, tumor-informed ctDNA assay was used to track a set of up to 16 somatic, single nucleotide variants in the associated patient’s plasma samples.ResultsPre-treatment ctDNA detection rate was 82% (14/17) and varied based on histology and stage. ctDNA was detected in 35% (6/17) of patients at the first post-RT timepoint (median of 1.66 months following the completion of RT), all of whom subsequently developed clinical progression. At this first post-RT time point, patients with ctDNA-positivity had significantly worse progression-free survival (PFS) [hazard ratio (HR): 24.2, p=0.004], and ctDNA-positivity was the only significant prognostic factor associated with PFS (HR: 13.4, p=0.02) in a multivariate analysis. All patients who developed clinical recurrence had detectable ctDNA with an average lead time over radiographic progression of 5.4 months, and post-RT ctDNA positivity was significantly associated with poor PFS (p<0.0001).ConclusionPersonalized, longitudinal ctDNA monitoring can detect recurrence early in patients with unresectable NSCLC patients undergoing curative radiation and potentially risk-stratify patients who might benefit most from treatment intensification

Acyl-isothiocyanates as efficient thiocyanate transfer reagents

An unprecedented transfer of a thiocyanate (−SCN) group from aroyl/acyl isothiocyanate to alkyl or benzylic bromide is observed in the presence of a tertiary amine. This process is most effective when the bromomethyl proton is less acidic, while the presence of a more acidic proton gives 1,3-oxathiol-2-ylidine and other related products

Positive predictive value of a single nucleotide polymorphism (SNP)‐based NIPT for aneuploidy in twins: Experience from clinical practice

ObjectiveTwins account for approximately 1 in 30 live births in the United States. However, there are limited clinical experience studies published in noninvasive prenatal testing (NIPT) for detecting aneuploidies in twins. This study reports the performance of an SNP-based NIPT in the largest cohort with known outcomes for high-risk aneuploidy results.MethodThis is a retrospective analysis of 18,984 results from commercial single-nucleotide polymorphism (SNP)-based NIPT tests performed in twins between October 2, 2017 and December 31, 2019. Follow-up for all 211 high-risk cases was solicited.ResultsFollow-up outcomes were obtained in 105 cases. Positive predictive values (PPVs) for high-risk results were 88.7% (63/71, 95% Confidence Interval [CI]: 79.0%-95.0%) for trisomy 21% and 72.7% (8/11, 95% CI: 39.0%-94.0%) for trisomy 18. The results were stratified into monozygotic (MZ) and dizygotic (DZ). The PPVs in MZ were 100% for both trisomy 21 (4/4, 95% CI: 40%-100%) and trisomy 18 (1/1, 95% CI: 2.5%-100%). No trisomy 13 cases were detected in the MZ group. The PPVs in DZ were 88.1% (59/67, 95% CI: 77.8%-94.7%), 70.0% (7/10, 95% CI: 34.8%-93.3%), and 66.7% (2/3, 95% CI: 9.4%-99.2%) for trisomy 21, trisomy 18, and trisomy 13, respectively.ConclusionThe performance of SNP-based NIPT in this large twin cohort was comparable to previously reported twin NIPT studies. SNP-based NIPT allows for zygosity-based PPV assessment

Nitrodibenzofuran: A One- and Two-Photon Sensitive Protecting Group That Is Superior to Brominated Hydroxycoumarin for Thiol Caging in Peptides

Photoremovable protecting groups are important for a wide range of applications in peptide chemistry. Using Fmoc-Cys­(Bhc-MOM)-OH, peptides containing a Bhc-protected cysteine residue can be easily prepared. However, such protected thiols can undergo isomerization to a dead-end product (a 4-methylcoumarin-3-yl thioether) upon photolysis. To circumvent that photoisomerization problem, we explored the use of nitrodibenzofuran (NDBF) for thiol protection by preparing cysteine-containing peptides where the thiol is masked with an NDBF group. This was accomplished by synthesizing Fmoc-Cys­(NDBF)-OH and incorporating that residue into peptides by standard solid-phase peptide synthesis procedures. Irradiation with 365 nm light or two-photon excitation with 800 nm light resulted in efficient deprotection. To probe biological utility, thiol group uncaging was carried out using a peptide derived from the protein K-Ras4B to yield a sequence that is a known substrate for protein farnesyltransferase; irradiation of the NDBF-caged peptide in the presence of the enzyme resulted in the formation of the farnesylated product. Additionally, incubation of human ovarian carcinoma (SKOV3) cells with an NDBF-caged version of a farnesylated peptide followed by UV irradiation resulted in migration of the peptide from the cytosol/Golgi to the plasma membrane due to enzymatic palmitoylation. Overall, the high cleavage efficiency devoid of side reactions and significant two-photon cross-section of NDBF render it superior to Bhc for thiol group caging. This protecting group should be useful for a plethora of applications ranging from the development of light-activatable cysteine-containing peptides to the development of light-sensitive biomaterials

Comprehensive Genomic Profiling (CGP)-Informed Personalized Molecular Residual Disease (MRD) Detection: An Exploratory Analysis from the PREDATOR Study of Metastatic Colorectal Cancer (mCRC) Patients Undergoing Surgical Resection

A majority of patients with metastatic colorectal cancer (mCRC) experience recurrence post curative-intent surgery. The addition of adjuvant chemotherapy has shown to provide limited survival benefits when applied to all patients. Therefore, a biomarker to assess molecular residual disease (MRD) accurately and guide treatment selection is highly desirable for high-risk patients. This feasibility study evaluated the prognostic value of a tissue comprehensive genomic profiling (CGP)-informed, personalized circulating tumor DNA (ctDNA) assay (FoundationOne®Tracker) (Foundation Medicine, Inc., Cambridge, MA, USA) by correlating MRD status with clinical outcomes. ctDNA analysis was performed retrospectively on plasma samples from 69 patients with resected mCRC obtained at the MRD and the follow-up time point. Tissue CGP identified potentially actionable alterations in 54% (37/69) of patients. MRD-positivity was significantly associated with lower disease-free survival (DFS) (HR: 4.97, 95% CI: 2.67–9.24, p < 0.0001) and overall survival (OS) (HR: 27.05, 95% CI: 3.60–203.46, p < 0.0001). Similarly, ctDNA positive status at the follow-up time point correlated with a marked reduction in DFS (HR: 8.78, 95% CI: 3.59–21.49, p < 0.0001) and OS (HR: 20.06, 95% CI: 2.51–160.25, p < 0.0001). The overall sensitivity and specificity at the follow-up time point were 69% and 100%, respectively. Our results indicate that MRD detection using the tissue CGP-informed ctDNA assay is prognostic of survival outcomes in patients with resected mCRC. The concurrent MRD detection and identification of actionable alterations has the potential to guide perioperative clinical decision-making

Metabolic Labeling with an Alkyne-modified Isoprenoid Analog Facilitates Imaging and Quantification of the Prenylome in Cells

Protein prenylation is a post-translational modification that is responsible for membrane association and protein–protein interactions. The oncogenic protein Ras, which is prenylated, has been the subject of intense study in the past 20 years as a therapeutic target. Several studies have shown a correlation between neurodegenerative diseases including Alzheimer’s disease and Parkinson’s disease and protein prenylation. Here, a method for imaging and quantification of the prenylome using microscopy and flow cytometry is described. We show that metabolically incorporating an alkyne isoprenoid into mammalian cells, followed by a Cu­(I)-catalyzed alkyne azide cycloaddition reaction to a fluorophore, allows for detection of prenylated proteins in several cell lines and that different cell types vary significantly in their levels of prenylated proteins. The addition of a prenyltransferase inhibitor or the precursors to the native isoprenoid substrates lowers the levels of labeled prenylated proteins. Finally, we demonstrate that there is a significantly higher (22%) level of prenylated proteins in a cellular model of compromised autophagy as compared to normal cells, supporting the hypothesis of a potential involvement of protein prenylation in abrogated autophagy. These results highlight the utility of total prenylome labeling for studies on the role of protein prenylation in various diseases including aging-related disorders