In this issue of Cell Chemical Biology, Chang et al. report that a PCNA-targeting bioPROTAC, an engineered E3 ligase linked to a PCNA-binding peptide, has superior pharmacological effects over ...inhibition of PCNA. Their findings demonstrate the feasibility of translating bioPROTACs from a concept to a candidate therapeutic modality.
In this issue of Cell Chemical Biology, Chang et al. report that a PCNA-targeting bioPROTAC, an engineered E3 ligase linked to a PCNA-binding peptide, has superior pharmacological effects over inhibition of PCNA. Their findings demonstrate the feasibility of translating bioPROTACs from a concept to a candidate therapeutic modality.
Trophoblast antigen 2 (Trop2) is a transmembrane glycoprotein upregulated in multiple solid tumours. Trop2-based passive immunotherapies are in clinical trials, while Trop2 targeting CAR-T cell-based ...therapies are also reported. Information about its T- and B-cell epitopes is needed for it to be pursued as an active immunotherapeutic target. This study focused on identification of immunodominant epitopes in the Trop2 extracellular domain (ECD) that can mount an efficient anti-Trop2 antibody response. In silico analysis using various B-cell epitope prediction tools was carried out to identify linear and conformational B-cell epitopes in the ECD of Trop2. Three linear peptide immunogens were shortlisted and synthesized. Along with linear peptides, truncated Trop2 ECD that possesses combination of linear and conformational epitopes was also selected. Recombinant protein immunogen was produced in 293-F suspension culture system and affinity purified. Antisera against different immunogens were characterized by ELISA and Western blotting. Two anti-peptide antisera detected recombinant and ectopically expressed Trop2 protein; however, they were unable to recognize the endogenous Trop2 protein expressed by cancer cells. Antibodies against truncated Trop2 ECD could bind to the endogenous Trop2 expressed on the surface of cancer cells. In addition to their high avidity, these polyclonal anti-sera against truncated Trop2 protein also mediated antibody-dependent cell-mediated cytotoxicity (ADCC). In summary, our comparative analysis demonstrated the utility of truncated Trop2 ECD as a promising candidate to be pursued as an active immunotherapeutic molecule against Trop2-positive cancer cells.
Proteolytic processing is an important post‐translational modification affecting protein activity and stability. In the current study, we investigate the N‐terminal cleavage of Trop2, a protein which ...is overexpressed in many cancers. We demonstrate that Trop2 is cleaved at Arg87 by a transmembrane serine protease, matriptase. Homology modeling and site‐directed mutagenesis of amino acids in close proximity to the matriptase cleavage site reveal the importance of Val194 in regulating Trop2 cleavage. Co‐immunoprecipitation studies confirm that amino acid substitutions at Arg87, Thr88, Lys189, Val194, and His195 do not affect Trop2 dimerization. However, cleavage of wild‐type Trop2 by matriptase is inhibited when it is allowed to dimerize with a V194A mutant monomer, further confirming the role of Val194 in matriptase‐mediated N‐terminal cleavage.
Trophoblast antigen 2 (Trop2) is a type I transmembrane protein post-translationally modified by N-linked glycosylation. It was originally detected in trophoblasts but was later shown to be ...frequently overexpressed in many epithelial cancers. Recently, anti-Trop2 antibody-drug conjugate has been FDA approved for the treatment of metastatic triple-negative breast and urothelial carcinomas, making it an important tumor antigen. The current study explored the significance of N-glycosylation of Trop2 by substituting specific N-glycan addition sites by site-directed mutagenesis. The mutant proteins were characterized in transiently transfected HEK293 cells. The N-glycosylation mutants did not affect protein expression, stability, dimerization ability and matriptase mediated cleavage. However, N120A and N208A mutants showed decreased interaction with its binding partner claudin-7. Our earlier reported Trop2 mutant V194A, which shows aberrant glycosylation, also displayed hampered interaction with claudin-7. To further characterize the mutants, stable clones expressing wild type and mutant Trop2 were generated in OVCAR3 cell line. Interestingly, surface biotinylation assay showed significantly higher surface expression of N120A and N208A mutants whereas surface localization was drastically reduced for V194A Trop2 mutant. Though overexpression of wild type Trop2 did not cause any change in fibronectin-mediated FAK (Focal adhesion kinase) signaling; expression of N120A mutant, surprisingly downregulated FAK signaling. Furthermore, exosomal release of Trop2 was also decreased in N120A and N208A mutants. This data suggests that site-specific N-glycan addition determines Trop2 surface density, claudin-7 interaction and exosomal release.
Android is a popular open-source operating system highly susceptible to malware attacks. Researchers have developed machine learning models, learned from attributes extracted using static/dynamic ...approaches to identify malicious applications. However, such models suffer from low detection accuracy, due to the presence of noisy attributes, extracted from conventional feature selection algorithms. Hence, in this paper, a new feature selection mechanism known as
selection of relevant attributes for improving locally extracted features using classical feature selectors
(SAILS), is proposed. SAILS, targets on discovering prominent system calls from applications, and is built on the top of conventional feature selection methods, such as mutual information, distinguishing feature selector and Galavotti–Sebastiani–Simi. These classical attribute selection methods are used as local feature selectors. Besides, a novel global feature selection method known as, weighted feature selection is proposed. Comprehensive analysis of the proposed feature selectors, is conducted with the traditional methods. SAILS results in improved values for evaluation metrics, compared to the conventional feature selection algorithms for distinct machine learning models, developed using Logistic Regression, CART, Random Forest, XGBoost and Deep Neural Networks. Our evaluations observe accuracies ranging between 95 and 99% for dropout rate and learning rate in the range 0.1–0.8 and 0.001–0.2, respectively. Finally, the security evaluation of malware classifiers on adversarial examples are thoroughly investigated. A decline in accuracy with adversarial examples is observed. Also, SAILS recall rate of classifier subjected to such examples estimate in the range of 24.79–92.2%. However, prior to the attack, the true positive rate obtained by the classifier is reported between 95.2 and 99.79%. The results suggest that the hackers can bypass detection, by discovering the classifier blind spots, on augmenting a small number of legitimate attributes.
The 2020 neonatal mortality rate in Bangladesh was 17 deaths per 1,000 live births, higher than the 2030 Sustainable Development Goal target (12 deaths per 1,000 live births). Over the past decade, ...Bangladesh has established special care newborn units (SCANUs) in medical facilities countrywide to improve neonatal survival. We conducted a retrospective cohort study in the SCANU of a tertiary-level healthcare facility in Bangladesh to study neonatal survival and associated risk factors using descriptive statistics and logistic regression models. Of 674 neonates admitted to the unit between January and November 2018, 263 (39%) died in hospital, 309 (46%) were discharged against medical advice, 90 (13%) were discharged healthy, and 12 (2%) had other discharge statuses. The median duration of time spent in hospital was 3 days, and 60% were admitted at birth. Neonates delivered via Cesarean section (adjusted odds ratio aOR: 2.5; 95% CI: 1.2-5.6) had an increased odds of recovering and being discharged, whereas those diagnosed with prematurity and/or low birth weight at admission (aOR: 0.2; 95% CI: 0.1-0.4) had a decreased odds of doing so. The high mortality rate and large number of neonates discharged against medical advice suggest a need to investigate the etiology of death and the factors leading to children leaving hospital prior to recovery. Medical records lacked information on gestational age that could provide key insights about mortality risk and age of viability in this setting. Addressing these knowledge gaps in SCANUs may enable better support for the improvement of child survival.
Patients with STEMI ( n = 385) had cardiac MRIs 2 to 3 days following reperfusion with primary PCI to determine the relationship between myocardial edema, LV mass, and MVO. We observed that MVO ...increased linearly with LV mass and that myocardial edema measured by T2-imaging also increased linearly with LV mass. Patients with MVO had greater edema and LVEDP than subjects without MVO. These findings suggest that myocardial edema which arises from ischemia-reperfusion injury may result in extravascular compression of the microcirculation manifested as MVO on cardiac MRI.
Microvascular obstruction (MVO) frequently develops after ST-elevation myocardial infarction (STEMI) and is associated with increased mortality and adverse left ventricular remodeling. We hypothesized that increased extravascular compressive forces in the myocardium that arise from the development of myocardial edema because of ischemia-reperfusion injury would contribute to the development of MVO. We measured MVO, infarct size, and left ventricular mass in patients with STEMI ( n = 385) using cardiac MRI 2 to 3 days following successful percutaneous coronary intervention and stenting. MVO was found in 57% of patients with STEMI. The average infarct size was 45 ± 29 g. Patients with MVO had significantly greater infarct size and reduced left ventricular (LV) function ( P < 0.01) compared with patients without MVO. Patients with MVO had significantly greater LV mass than patients without MVO and there was a linear increase in MVO with increasing LV mass ( P < 0.001). Myocardial edema by T2-weighted imaging increased with increasing LV mass and patients with MVO had significantly greater myocardial edema than patients without MVO ( P < 0.01). Patients with MVO had significantly greater left ventricular end-diastolic pressure (LVEDP) than patients without MVO ( P < 0.05). In a cohort of patients with STEMI who underwent primary percutaneous intervention, we observed that MVO increased linearly with increasing LV mass and was associated with increased myocardial edema and higher LVEDP. These observations support the concept that extravascular compressive forces in the left ventricle may increase with increasing ischemic injury and contribute to the development of MVO.
NEW & NOTEWORTHY Patients with STEMI ( n = 385) had cardiac MRIs 2 to 3 days following reperfusion with primary PCI to determine the relationship between myocardial edema, LV mass, and MVO. We observed that MVO increased linearly with LV mass and that myocardial edema measured by T2-imaging also increased linearly with LV mass. Patients with MVO had greater edema and LVEDP than subjects without MVO. These findings suggest that myocardial edema which arises from ischemia-reperfusion injury may result in extravascular compression of the microcirculation manifested as MVO on cardiac MRI.
•Three overlapping fragments of human CRISP3 promoter were cloned and characterized for promoter activity.•CRISP3 promoter construct showed highest activity in LNCaP stable clones as compared to PC3 ...stable clones.•Endogenous CRISP-3 expression was responsive to DHT in LNCaP.•CRISP-3 promoter showed histone H3 acetylation in LNCaP but not in PC3 cells.•AR occupied CRISP-3 promoter in LNCaP cells and was associated with acetylated histone H3.
Cysteine-rich secretory protein 3 (CRISP3) is one of the most upregulated genes in prostate cancer. Androgen receptor (AR) plays an important role not only in initial stages of prostate cancer development but also in the advanced stage of castration-resistant prostate cancer (CRPC). Role of AR in regulation of CRISP3 expression is not yet known. In order to understand the regulation of CRISP3 expression, various overlapping fragments of CRISP3 promoter were cloned in pGL3 luciferase reporter vector. All constructs were transiently and stably transfected in PC3 (CRISP3 negative) and LNCaP (CRISP3 positive) cell lines and promoter activity was measured by luciferase assay. Promoter activity of LNCaP stable clones was significantly higher than PC3 stable clones. Further in CRISP3 negative PC3 and RWPE-1 cells, CRISP3 promoter was shown to be silenced by histone deacetylation. Treatment of LNCaP cells with DHT resulted in increase in levels of CRISP3 transcript and protein. AR dependency of CRISP3 promoter was also evaluated in LNCaP stable clones by luciferase assay. To provide molecular evidence of epigenetic regulation of CRISP3 promoter and its response to DHT, ChIP PCR was performed in PC3 and LNCaP cells. Our results demonstrate that CRISP3 expression in prostate cancer cells is androgen dependent and in AR positive cells, CRISP3 promoter is epigenetically regulated by AR.
Background
Ovarian cancer is usually detected at an advanced stage with frequent recurrence. The recurrence-free survival and overall survival is influenced by the age at diagnosis, tumor stage and ...histological subtype. Nonetheless, quantifiable prognostic biomarkers are needed for early identification of the high-risk patients and for personalized medicine. Several studies link tumor-specific dysregulated expression of certain proteins with ovarian cancer prognosis. However, careful investigation of presence of these prognostically relevant proteins in ovarian cancer secretome is lacking.
Objective
To critically analyze the recent published data on prognostically relevant proteins for ovarian cancer and to carefully search how many of them are reported in the published ovarian cancer secretome datasets.
Design
A search for relevant studies in the past 2 years was conducted in PubMed and a comprehensive list of proteins associated with the ovarian cancer prognosis was prepared. These were cross-referred to the published ovarian cancer secretome profiles. The proteins identified in the secretome were further shortlisted based on a scoring strategy employing stringent criteria.
Results
A panel of seven promising secretory biomarkers associated with ovarian cancer prognosis is proposed.
Conclusion
Scanning the ovarian cancer secretome datasets provides the opportunity to identify if tumor-specific biomarkers could be tested as secretory biomarkers. Detecting their levels in the body fluid would be more advantageous than evaluating the expression in the tissue, since it could be monitored multiple times over the course of the disease to have a better judgment of the prognosis and response to therapy.
Background
Mammalian cysteine‐rich secretory proteins (CRISPs) are predominantly expressed in the male reproductive tract. Knockout mice lacking two or more CRISPs show defects in sperm transport, ...sperm–egg interaction and Ca2+ homeostasis. CRISPs play redundant and specific roles via their binding partners. To understand this, a comprehensive analysis of CRISP interactome needs to be undertaken.
Objectives
This study aimed to analyse CRISP4 binding partners on the plasma membrane of rat caudal spermatozoa.
Materials and methods
Total proteins from rat caudal spermatozoa were subjected to immunoprecipitation using anti‐CRISP4 antibody followed by liquid chromatography–mass spectrophotometry analysis. Plasma membrane localised proteins were shortlisted, and a key target was validated by co‐immunoprecipitation and co‐localisation. Co‐transfection followed by co‐immunoprecipitation was carried out for studying the interaction of full‐length as well as deletion mutants of CRISPs with human plasma membrane calcium ATPase, isoform b (hPMCA4b). Calcium assays were performed using Fura‐2‐AM. The cholesterol binding ability of different CRISPs was evaluated in silico.
Results
The membrane‐specific interactome of rat CRISP4 (rCRISP4) from caudal spermatozoa revealed PMCA4b as a novel binding partner, and their interaction was validated in rat spermatozoa. Human CRISP1 (hCRISP1) and hCRISP3 also interacted with PMCA4b via the N‐terminal domain. Interestingly, hCRISP1 and rCRISP4 delayed PMCA4b‐mediated calcium extrusion but hCRISP3 did not. In silico analysis demonstrated that hCRISP1 and rCRISP4 have higher binding affinity towards cholesterol than hCRISP3. The secretion profile of different CRISPs also showed that the ratio of secreted to cell‐associated proteins was highest for hCRISP3.
Conclusion
Our study identifies PMCA4b as a target of multiple mammalian CRISPs and unravels a new role of CRISPs in regulating calcium homeostasis. Differences in the interaction of different CRISPs with cholesterol may regulate their enrichment in the lipid rafts and redistribution in the membrane post‐capacitation, thereby affecting their interaction with PMCA4b.
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