Multiple circulating forms of neprilysin detected with novel epitope-directed monoclonal antibodies.

Neprilysin (NEP) is an emerging biomarker for various diseases including heart failure (HF). However, major inter-assay inconsistency in the reported concentrations of circulating NEP and uncertainty with respect to its correlations with type and severity of disease are in part attributed to poorly characterized antibodies supplied in commercial ELISA kits. Validated antibodies with well-defined binding footprints are critical for understanding the biological and clinical context of NEP immunoassay data. To achieve this, we applied in silico epitope prediction and rational peptide selection to generate monoclonal antibodies (mAbs) against spatially distant sites on NEP. One of the selected epitopes contained published N-linked glycosylation sites at N285 and N294. The best antibody pair, mAb 17E11 and 31E1 (glycosylation-sensitive), were characterized by surface plasmon resonance, isotyping, epitope mapping, and western blotting. A validated two-site sandwich NEP ELISA with a limit of detection of 2.15 pg/ml and working range of 13.1-8000 pg/ml was developed with these mAbs. Western analysis using a validated commercial polyclonal antibody (PE pAb) and our mAbs revealed that non-HF and HF plasma NEP circulates as a heterogenous mix of moieties that possibly reflect proteolytic processing, post-translational modifications and homo-dimerization. Both our mAbs detected a ~ 33 kDa NEP fragment which was not apparent with PE pAb, as well as a common ~ 57-60 kDa moiety. These antibodies exhibit different affinities for the various NEP targets. Immunoassay results are dependent on NEP epitopes variably detected by the antibody pairs used, explaining the current discordant NEP measurements derived from different ELISA kits.

Is Short-Course Antibiotic Therapy Suitable for Pseudomonas aeruginosa Bloodstream Infections in Onco-hematology Patients With Febrile Neutropenia? Results of a Multi-institutional Analysis.

BACKGROUND: Several studies have suggested that short-course antibiotic therapy was effective in Pseudomonas aeruginosa (PA) bloodstream infections (BSI) in immunocompetent patients. But similar studies in patients with hematological malignancies were rare. METHODS: This cohort study included onco-hematology patients at 2 hematology centers in China. Inverse probability of treatment weighting was used to balance the confounding factors. Multivariate regression model was used to evaluate the effect of short-course antibiotic therapy on clinical outcomes. RESULTS: In total, 434 patients met eligibility criteria (short-course, 7-11 days, n = 229; prolonged, 12-21 days, n = 205). In the weighted cohort, the univariate and multivariate analysis indicated that short course antibiotic therapy had similar outcomes to the prolonged course. The recurrent PA infection at any site or mortality within 30 days of completing therapy occurred in 8 (3.9%) patients in the short-course group and in 10 (4.9%) in the prolonged-course group (P = .979). The recurrent infection within 90 days occurred in 20 (9.8%) patients in the short-course group and in 13 (6.3%) patients in the prolonged-course group (P = .139), and the recurrent fever within 7 days occurred in 17 (8.3%) patients in the short-course group and in 15 (7.4%) in the prolonged-course group (P = .957). On average, patients who received short-course antibiotic therapy spent 3.3 fewer days in the hospital (P < .001). CONCLUSIONS: In the study, short-course therapy was non-inferior to prolonged-course therapy in terms of clinical outcomes. However, due to its biases and limitations, further prospective randomized controlled trials are needed to generalize our findings.

Dynamic altruistic cooperation within breast tumors.

BACKGROUND: Social behaviors such as altruism, where one self-sacrifices for collective benefits, critically influence an organism's survival and responses to the environment. Such behaviors are widely exemplified in nature but have been underexplored in cancer cells which are conventionally seen as selfish competitive players. This multidisciplinary study explores altruism and its mechanism in breast cancer cells and its contribution to chemoresistance. METHODS: MicroRNA profiling was performed on circulating tumor cells collected from the blood of treated breast cancer patients. Cancer cell lines ectopically expressing candidate miRNA were used in co-culture experiments and treated with docetaxel. Ecological parameters like relative survival and relative fitness were assessed using flow cytometry. Functional studies and characterization performed in vitro and in vivo include proliferation, iTRAQ-mass spectrometry, RNA sequencing, inhibition by small molecules and antibodies, siRNA knockdown, CRISPR/dCas9 inhibition and fluorescence imaging of promoter reporter-expressing cells. Mathematical modeling based on evolutionary game theory was performed to simulate spatial organization of cancer cells. RESULTS: Opposing cancer processes underlie altruism: an oncogenic process involving secretion of IGFBP2 and CCL28 by the altruists to induce survival benefits in neighboring cells under taxane exposure, and a self-sacrificial tumor suppressive process impeding proliferation of altruists via cell cycle arrest. Both processes are regulated concurrently in the altruists by miR-125b, via differential NF-κB signaling specifically through IKKß. Altruistic cells persist in the tumor despite their self-sacrifice, as they can regenerate epigenetically from non-altruists via a KLF2/PCAF-mediated mechanism. The altruists maintain a sparse spatial organization by inhibiting surrounding cells from adopting the altruistic fate via a lateral inhibition mechanism involving a GAB1-PI3K-AKT-miR-125b signaling circuit. CONCLUSIONS: Our data reveal molecular mechanisms underlying manifestation, persistence and spatial spread of cancer cell altruism. A minor population behave altruistically at a cost to itself producing a collective benefit for the tumor, suggesting tumors to be dynamic social systems governed by the same rules of cooperation in social organisms. Understanding cancer cell altruism may lead to more holistic models of tumor evolution and drug response, as well as therapeutic paradigms that account for social interactions. Cancer cells constitute tractable experimental models for fields beyond oncology, like evolutionary ecology and game theory.

The role of acute normovolemic hemodilution in reducing allogeneic blood transfusion in glioblastoma surgery: a case-control study.

BACKGROUND: Acute normovolemic hemodilution (ANH) was first introduced in glioblastoma surgery, and its role in reducing allogeneic blood transfusion was investigated in this study. METHODS: This study enrolled supratentorial glioblastoma patients who received total resection. In the ANH group, the patients were required to draw blood before the operation, and the blood will be transfused back to the patient during the operation. The association between ANH and clinical features was investigated. RESULTS: Sixty supratentorial glioblastoma patients were enrolled in this study, 25 patients were allocated in the ANH group, and another 35 patients were included in the control group. ANH dramatically reduced the need for allogeneic blood transfusion (3 [12%] vs 12 [34.3%], P = 0.049), and the blood transfusion per total of patients was dramatically decreased by the application of ANH (0.40 ± 1.15 units vs 1.06 ± 1.59 units, P = 0.069). Furthermore, ANH also markedly reduced the requirement of fresh frozen plasma (FFP) transfusion (2 [8%] vs 11 [31.4%], P = 0.030) and the volume of FFP transfusion per total of patients (32.00 ± 114.46 mL vs 115.71 ± 181.00 mL, P = 0.033). The complication rate was similar between the two groups. CONCLUSIONS: ANH was a safe and effective blood conservation technique in glioblastoma surgery.

Quantitative Chemical Proteomics Reveals Triptolide Selectively Inhibits HCT116 Human Colon Cancer Cell Viability and Migration Through Binding to Peroxiredoxin 1 and Annexin A1.

Triptolide (TPL), a natural product extracted from Tripterygium wilfordii Hook F, exerts potential anti-cancer activity. Studies have shown that TPL is involved in multiple cellular processes and signal pathways; however, its pharmaceutical activity in human colorectal cancer (CRC) as well as the underlying molecular mechanism remain elusive. In this study, the effects of TPL on HCT116 human colon cancer cells and CCD841 human colon epithelial cells are first evaluated. Next, the protein targets of TPL in HCT116 cells are identified through an activity-based protein profiling approach. With subsequent in vitro experiments, the mode of action of TPL in HCT116 cells is elucidated. As a result, TPL is found to selectively inhibit HCT116 cell viability and migration. A total of 54 proteins are identified as the targets of TPL in HCT116 cells, among which, Annexin A1 (ANXA1) and Peroxiredoxin I/II (Prdx I/II) are picked out for further investigation due to their important role in CRC. The interaction between TPL and ANXA1 or Prdx I is confirmed, and it is discovered that TPL exerts inhibitory effect against HCT116 cells through binding to ANXA1 and Prdx I. The study reinforces the potential of TPL in the CRC therapy, and provides novel therapeutic targets for the treatment of CRC.

Sequential genome-wide CRISPR-Cas9 screens identify genes regulating cell-surface expression of tetraspanins.

Tetraspanins, a superfamily of membrane proteins, mediate diverse biological processes through tetraspanin-enriched microdomains in the plasma membrane. However, how their cell-surface presentation is controlled remains unclear. To identify the regulators of tetraspanin trafficking, we conduct sequential genome-wide loss-of-function CRISPR-Cas9 screens based on cell-surface expression of a tetraspanin member, TSPAN8. Several genes potentially involved in endoplasmic reticulum (ER) targeting, different biological processes in the Golgi apparatus, and protein trafficking are identified and functionally validated. Importantly, we find that biantennary N-glycans generated by MGAT1/2, but not more complex glycan structures, are important for cell-surface tetraspanin expression. Moreover, we unravel that SPPL3, a Golgi intramembrane-cleaving protease reported previously to act as a sheddase of multiple glycan-modifying enzymes, controls cell-surface tetraspanin expression through a mechanism associated with lacto-series glycolipid biosynthesis. Our study provides critical insights into the molecular regulation of cell-surface presentation of tetraspanins with implications for strategies to manipulate their functions, including cancer cell invasion.

SFPQ promotes RAS-mutant cancer cell growth by modulating 5'-UTR mediated translational control of CK1α.

Oncogenic mutations in the RAS family of small GTPases are commonly found in human cancers and they promote tumorigenesis by altering gene expression networks. We previously demonstrated that Casein Kinase 1α (CK1α), a member of the CK1 family of serine/threonine kinases, is post-transcriptionally upregulated by oncogenic RAS signaling. Here, we report that the CK1α mRNA contains an exceptionally long 5'-untranslated region (UTR) harbouring several translational control elements, implicating its involvement in translational regulation. We demonstrate that the CK1α 5'-UTR functions as an IRES element in HCT-116 colon cancer cells to promote cap-independent translation. Using tobramycin-affinity RNA-pulldown assays coupled with identification via mass spectrometry, we identified several CK1α 5'-UTR-binding proteins, including SFPQ. We show that RNA interference targeting SFPQ reduced CK1α protein abundance and partially blocked RAS-mutant colon cancer cell growth. Importantly, transcript and protein levels of SFPQ and other CK1α 5'-UTR-associated RNA-binding proteins (RBPs) are found to be elevated in early stages of RAS-mutant cancers, including colorectal and lung adenocarcinoma. Taken together, our study uncovers a previously unappreciated role of RBPs in promoting RAS-mutant cancer cell growth and their potential to serve as promising biomarkers as well as tractable therapeutic targets in cancers driven by oncogenic RAS.

Complicated multiple organ infection of Purpureocillium lilacinum and varicella-zoster virus infection in a patient with Evans' syndrome.

Purpureocillium lilacinum (P lilacinum) is a rare pathogenic fungus, which mainly involves immunocompromised individuals. Here, we report a case of complicated multiple-organ infections involving skin, lungs, and spleen in a 63-year-old female with Evans' syndrome after 9 months of glucocorticoid treatment. Microbial examinations of skin biopsy and blood samples revealed P lilacinum infections. Posaconazole was effective in this patient. During anti-fungi treatment, she developed varicella-zoster virus infection and was diagnosed through next-generation sequencing examination. In conclusion, P lilacinum may affect different organ systems and is susceptible to posaconazole treatment. The molecular-based methods like microbial cell-free DNA sequencing could provide accurate and timely identification of a wide range of infections.

Selective Inhibition of Plasmodium falciparum ATPase 6 by Artemisinins and Identification of New Classes of Inhibitors after Expression in Yeast.

Treatment failures with artemisinin combination therapies (ACTs) threaten global efforts to eradicate malaria. They highlight the importance of identifying drug targets and new inhibitors and of studying how existing antimalarial classes work. Here, we report the successful development of a heterologous expression-based compound-screening tool. The validated drug target Plasmodium falciparum ATPase 6 (PfATP6) and a mammalian orthologue (sarco/endoplasmic reticulum calcium ATPase 1a [SERCA1a]) were functionally expressed in Saccharomyces cerevisiae, providing a robust, sensitive, and specific screening tool. Whole-cell and in vitro assays consistently demonstrated inhibition and labeling of PfATP6 by artemisinins. Mutations in PfATP6 resulted in fitness costs that were ameliorated in the presence of artemisinin derivatives when studied in the yeast model. As previously hypothesized, PfATP6 is a target of artemisinins. Mammalian SERCA1a can be mutated to become more susceptible to artemisinins. The inexpensive, low-technology yeast screening platform has identified unrelated classes of druggable PfATP6 inhibitors. Resistance to artemisinins may depend on mechanisms that can concomitantly address multitargeting by artemisinins and fitness costs of mutations that reduce artemisinin susceptibility.

Quantitative chemical proteomics reveals anti-cancer targets of Celastrol in HCT116 human colon cancer cells.

BACKGROUND: Celastrol (Cel) is a naturally-derived compound with anti-cancer properties and exerts beneficial effects against various diseases. Although an extensive body of research already exists for Cel, the vast majority are inductive studies with limited validation of specific pathways and functions. The cellular targets that bind to Cel remain poorly characterized, which limits attempts to uncover its mechanism of action. PURPOSE: The present study aims to comprehensively identify the protein targets of Cel in HCT116 cells in an unbiased manner, and elucidate the mechanism of the anti-cancer activity of Cel based on target information. METHODS: A comprehensive analysis of protein targets that bind to Cel was performed in HCT116 colon cancer cells using a quantitative chemical biology method. A Cel probe (Cel-P) was synthesized to allow in situ monitoring of treatment in living HCT116 cells, and specific targets were identified with a quantitative chemical biology method (isobaric tags for relative and absolute quantitation) using mass spectrometry. RESULTS: In total, 100 protein targets were identified as specific targets of Cel. Pathways associated with the targets were investigated. Multiple pathways were demonstrated to be potential effectors of Cel. These pathways included the suppression of protein synthesis, deregulation of cellular reactive oxygen species, and suppression of fatty acid metabolism, and they were validated with in vitro experiments. CONCLUSION: The extensive information on the protein targets of Cel and their functions uncovered by this study will enhance the current understanding of the mechanism of action of Cel and serve as a valuable knowledge base for future studies.

Finding a reliable assay for soluble neprilysin.

BACKGROUND: Lack of validation and standardization of research-use-only (RUO) immunoassays brings with it inherent threats to authenticity and functional quality. Poor correlation between different commercial neprilysin RUO immunoassays is concerning and discordant findings need to be resolved. We seek to identify and validate reliable neprilysin immunoassays to strengthen the scientific rigor and reproducibility of neprilysin-related investigation and of biomarker research in general. METHODS: Soluble neprilysin (sNEP) concentrations were determined in cohorts (n = 532) from Spain (Cohort 1), New Zealand (NZ, Cohort 2) and Singapore (Cohort 3), using commercial kits from six vendors. Apparent sNEP concentrations were correlated between different assays and with plasma neprilysin activity. Assay reliability was further validated by performance verification, MS analysis and cross-reactivity tests. RESULTS: sNEP in Cohorts 1 and 2 measured concurrently in Spain and NZ showed significant inter-laboratory correlation only for the Aviscera Bioscience sNEP ELISA SK00724-01. Neprilysin concentrations obtained with the R&D systems and SK00724-01 ELISAs correlated with each other but not with neprilysin activity. In Cohort 3, sNEP concentrations from the Perkin Elmer AlphaLISA and Biotechne ELLA assays agreed (r = 0.89) and both correlated with neprilysin activity (r = 0.87, 0.77 respectively). MS analysis detected authentic neprilysin in the AlphaLISA kit calibrator and in antibody pull-down material from human plasma. The AlphaLISA assay performed within acceptable limits (spike and recovery, dilutional linearity, inter- and intra-assay CV) and showed no cross-reactivity against neprilysin substrates and closely-related analogues. CONCLUSION: AlphaLISA and ELLA assays provide reliable measures of sNEP concentrations. Reliability of other commercial neprilysin assays remains in question.

O-GlcNAcylation promotes fatty acid synthase activity under nutritional stress as a pro-survival mechanism in cancer cells.

Protein O-GlcNAcylation is a specific form of protein glycosylation that targets a wide range of proteins with important functions. O-GlcNAcylation is known to be deregulated in cancer and has been linked to multiple aspects of cancer pathology. Despite its ubiquity and importance, the current understanding of the role of O-GlcNAcylation in the stress response remains limited. In this study, we performed a quantitative chemical proteomics-based open study of the O-GlcNAcome in HeLa cells, and identified 163 differentially-glycosylated proteins under starvation, involving multiple metabolic pathways. Among them, fatty acid metabolism was found to be targeted and subsequent analysis confirmed that fatty acid synthase (FASN) is O-GlcNAcylated. O-GlcNAcylation led to enhanced de novo fatty acid synthesis (FAS) activity, and fatty acids contributed to the cytoprotective effects of O-GlcNAcylation under starvation. Moreover, dual inhibition of O-GlcNAcylation and FASN displayed a strong synergistic effect in vitro in inducing cell death in cancer cells. Together, the results from this study provide novel insights into the role of O-GlcNAcylation in the nutritional stress response and suggest the potential of combining inhibition of O-GlcNAcylation and FAS in cancer therapy.

Fumarase affects the deoxyribonucleic acid damage response by protecting the mitochondrial desulfurase Nfs1p from modification and inactivation.

The Krebs cycle enzyme fumarase, which has been identified as a tumor suppressor, is involved in the deoxyribonucleic acid (DNA) damage response (DDR) in human, yeast, and bacterial cells. We have found that the overexpression of the cysteine desulfurase Nfs1p restores DNA repair in fumarase-deficient yeast cells. Nfs1p accumulates inactivating post-translational modifications in yeast cells lacking fumarase under conditions of DNA damage. Our model is that in addition to metabolic signaling of the DDR in the nucleus, fumarase affects the DDR by protecting the desulfurase Nfs1p in mitochondria from modification and inactivation. Fumarase performs this protection by directly binding to Nfs1p in mitochondria and enabling, the maintenance, via metabolism, of a non-oxidizing environment in mitochondria. Nfs1p is required for the formation of Fe-S clusters, which are essential cofactors for DNA repair enzymes. Thus, we propose that the overexpression of Nfs1p overcomes the lack of fumarase by enhancing the activity of DNA repair enzymes.

Isobaric tags for relative and absolute quantification (iTRAQ)-based quantitative analysis of the salivary proteome during healthy pregnancy and pregnancy gingivitis.

AIM: The present study aimed to investigate the salivary proteome profiles of pregnant women with gingivitis (PG) or without gingivitis (HP) and non-pregnant healthy controls (HC) by employing iTRAQ-based proteomics. MATERIALS AND METHODS: Saliva samples were collected from 30 Chinese women comprising 10 subjects in each of the three groups (PG, HP, and HC). The samples were subjected to iTRAQ-based proteomics analysis, and ELISA was performed to validate the results. The subsequent observations were validated in a cohort of 48 subjects. RESULTS: Pathways associated with neutrophil-mediated immune response and antioxidant defence mechanism were significantly higher in PG than HC. The abundance of salivary cystatins (S, SA, and SN) and antimicrobials were significantly decreased in PG and HP, while cystatin C and D were additionally decreased in PG. Cystatin C was mapped to all the major catabolic pathways and was the most re-wired protein in pregnancy gingivitis. Further validation demonstrated cystatin C to be significantly lower in PG than HC. CONCLUSIONS: While the decrease in levels of salivary cystatins and antimicrobial proteins may predispose healthy pregnant women to pregnancy gingivitis, it may cause persistence of inflammation in pregnant women with gingivitis.

Epitope-directed monoclonal antibody production using a mixed antigen cocktail facilitates antibody characterization and validation.

High quality, well-validated antibodies are needed to mitigate irreproducibility and clarify conflicting data in science. We describe an epitope-directed monoclonal antibody (mAb) production method that addresses issues of antibody quality, validation and utility. The workflow is illustrated by generating mAbs against multiple in silico-predicted epitopes on human ankyrin repeat domain 1 (hANKRD1) in a single hybridoma production cycle. Antigenic peptides (13-24 residues long) presented as three-copy inserts on the surface exposed loop of a thioredoxin carrier produced high affinity mAbs that are reactive to native and denatured hANKRD1. ELISA assay miniaturization afforded by novel DEXT microplates allowed rapid hybridoma screening with concomitant epitope identification. Antibodies against spatially distant sites on hANKRD1 facilitated validation schemes applicable to two-site ELISA, western blotting and immunocytochemistry. The use of short antigenic peptides of known sequence facilitated direct epitope mapping crucial for antibody characterization. This robust method motivates its ready adoption for other protein targets.

CSF-CN contributes to cancer-induced bone pain via the MKP-1-mediated MAPK pathway.

Pain is a major complication of cancer and significantly affects the quality of life. Cerebrospinal fluid-contacting nucleus (CSF-CN) has been reported to be involved in the development of neuropathic pain and inflammatory pain. However, whether CSF-CN contributes to cancer-induced bone pain (CIBP) remains unknown. In this study, we aimed to illustrate the role of CSF-CN in the pathogenesis of CIBP and identify its potential mechanism via the MKP-1-mediated MAPK pathway. The Walker 256 cancer cells were injected into the tibia cavity of female Sprague-Dawley rats to induce CIBP models. Intracerebroventricular injection of cholera toxin subunit B- saporin (CB-SAP) was performed to "knockout" the CSF-CN. Morphine and LV-MKP-1 were applied. Mechanical and thermal hyperalgesia behaviors, double immunofluorescence staining and Western blot were conducted after CIBP induction. The results revealed that CIBP significantly reduced the mechanical withdrawal threshold and the thermal threshold. Double immunofluorescence staining revealed that c-Fos-positive neurons in CSF-CN were significantly higher in the CIBP group than that in the sham group. Targeted ablation of CSF-CN dramatically aggravated pain sensitivity. Moreover, MKP-1 was down-regulated in the CSF-CN after CIBP induction. Pharmacological intervention with morphine significantly ameliorated the mechanical and thermal hyperalgesia through reversing the down-expression of MKP-1 in the CSF-CN on day 14 after CIBP induction. Mechanically, overexpression of MKP-1 by LV-MKP-1 injection significantly relieved CIBP via inhibiting the expression of phosphorylated p38, which subsequently decreased the protein levels of Bax, cleaved caspase-3 and Iba-1, and reduced the mRNA levels of IL-1ß, TNF-α and IL-6 in CSF-CN. In conclusion, CSF-CN contributed to CIBP via regulating the MKP-1-mediated p38-MAPK pathway. Future therapy targeting the expression of MKP-1 in the CSF-CN may be a promising new choice.

The Use of Acute Normovolemic Hemodilution in Clipping Surgery for Aneurysmal Subarachnoid Hemorrhage.

BACKGROUND: The occurrence of coronavirus disease 2019 (COVID-19) has overwhelmed the blood supply chain worldwide and severely influenced clinical procedures with potential massive blood loss, such as clipping surgery for aneurysmal subarachnoid hemorrhage (aSAH). Whether acute normovolemic hemodilution (ANH) is safe and effective in aneurysm clipping remains largely unknown. METHODS: Patients with aSAH who underwent clipping surgery within 72 hours from bleeding were included. The patients in the ANH group received 400 mL autologous blood collection, and the blood was returned as needed during surgery. The relationships between ANH and perioperative allogeneic blood transfusion, postoperative outcome, and complications were analyzed. RESULTS: Sixty-two patients with aSAH were included between December 2019 and June 2020 (20 in the ANH group and 42 in the non-ANH group). ANH did not reduce the need of perioperative blood transfusion (3 [15%] vs. 5 [11.9%]; P = 0.734). However, ANH significantly increased serum hemoglobin levels on postoperative day 1 (11.5 ± 2.5 g/dL vs. 10.3 ± 2.0 g/dL; P = 0.045) and day 3 (12.1 ± 2.0 g/dL vs. 10.7 ± 1.3 g/dL; P = 0.002). Multivariable analysis indicated that serum hemoglobin level on postoperative day 1 (odds ratio, 0.895; 95% confidence interval, 0.822-0.973; P = 0.010) was an independent risk factor for unfavorable outcome, and receiver operating characteristic curve analysis showed that it had a comparable predictive power to World Federation of Neurosurgical Societies grade (Z = 0.275; P > 0.05). CONCLUSIONS: ANH significantly increased postoperative hemoglobin levels, and it may hold the potential to improve patients' outcomes. Routine use of ANH should be considered in aneurysm clipping surgery.

Target Profiling of an Anticancer Drug Curcumin by an In Situ Chemical Proteomics Approach.

Interdisciplinary chemical proteomics approaches have been widely applied to the identification of specific targets of bioactive small molecules or drugs. In this chapter, we describe the application of a cell-permeable activity-based curcumin probe (Cur-P) with an alkyne moiety to detect and identify specific binding targets of curcumin in HCT116 colon cancer cells. Through click chemistry, a fluorescent tag or a biotin tag is attached to the probe-modified curcumin targets for visualization or affinity purification followed by mass spectrometric identification. A quantitative proteomics approach of isobaric tags for relative and absolute quantification (iTRAQ)™ is applied to distinguish specific curcumin targets from nonspecific binding proteins.

Risk Factors and Outcomes of Antibiotic-resistant Pseudomonas aeruginosa Bloodstream Infection in Adult Patients With Acute Leukemia.

BACKGROUND: Pseudomonas aeruginosa (PA) bloodstream infection (BSI) is a common complication in patients with acute leukemia (AL), and the prevalence of antibiotic-resistant strains poses a serious problem. However, there is limited information regarding antibiotic resistance, clinical characteristics, and outcomes of PA BSI in AL patients. This study explored characteristics associated with the clinical outcomes of AL patients with PA BSI and analyzed factors associated with BSI caused by multidrug-resistant (MDR) or carbapenem-resistant strains. METHODS: This single-center retrospective study enrolled hospitalized AL patients who developed PA BSI during January 2014-December 2019. The Kaplan-Meier method was used to plot survival curves. Multivariate logistic regression analyses were also performed. RESULTS: Of 293 eligible patients with PA BSI, 55 (18.8%) received inappropriate empirical antibiotic therapy within 48 hours of BSI onset, whereas up to 65.8% MDR-PA BSI patients received inappropriate empirical treatment. The 30-day mortality rate was 8.5% for all patients. However, the 30-day mortality rates were 28.9% and 5.5% in MDR-PA BSI and non-MDR-PA BSI patients, respectively (P < .001). On multivariate analysis, previous use of quinolones (odds ratio [OR], 5.851 [95% confidence interval {CI}, 2.638-12.975]) and piperacillin/tazobactam (OR, 2.837 [95% CI, 1.151-6.994]) were independently associated with MDR-PA BSI; and MDR-PA BSI (OR, 7.196 [95% CI, 2.773-18.668]), perianal infection (OR, 4.079 [95% CI, 1.401-11.879]), pulmonary infection (OR, 3.028 [95% CI, 1.231-7.446]), and age ≥55 years (OR, 2.871 [95% CI, 1.057-7.799]) were independent risk factors for 30-day mortality. CONCLUSIONS: MDR increases mortality risk in PA BSI patients, and previous antibiotic exposure is important in MDR-PA BSI development. Rational antibiotic use based on local antimicrobial susceptibility and clinical characteristics can help reduce antibiotic resistance and mortality.