Translocation of Non-Canonical Polypeptides into Cells Using Protective Antigen

A variety of pathogenic bacteria infect host eukaryotic cells using protein toxins, which enter the cytosol and exert their cytotoxic effects. Anthrax lethal toxin, for example, utilizes the membrane-spanning translocase, protective antigen (PA) pore, to deliver the protein toxin lethal factor (LF) from the endosome into the cytosol of cells. Previous work has investigated the delivery of natural peptides and enzymatic domains appended to the C-terminus of the PA-binding domain of lethal factor (LF[subscript N]) into the cytosol via PA pore. Here, we move beyond natural amino acids and systematically investigate the translocation of polypeptide cargo containing non-canonical amino acids and functionalities through PA pore. Our results indicate translocation is not perturbed with alterations to the peptide backbone or side-chain. Moreover, despite their structural complexity, we found that the small molecule drugs, doxorubicin and monomethyl auristatin F (MMAF) translocated efficiently through PA pore. However, we found cyclic peptides and the small molecule drug docetaxel abrogated translocation due to their large size and structural rigidity. For cargos that reached the cytosol, we demonstrated that each remained intact after translocation. These studies show PA is capable of translocating non-canonical cargo provided it is in a conformational state conducive for passage through the narrow pore.MIT Start-up FundsMassachusetts Institute of Technology. Charles E. Reed Faculty Initiative FundDamon Runyon Cancer Research Foundation (Innovation Award)National Science Foundation (U.S.) (CAREER Award CHE-1351807)National Science Foundation (U.S.). Graduate Research Fellowshi

Serum Amyloid A and Lipoprotein Associated Phospholipase A(2) Levels in Patients with Malign Melanoma: Correlations with Clinical Assessment and Stage

WOS: 000443333400004Objective: The lack of validated, sensitive, and specific biomarkers for early diagnosis and follow-up of patients with malign melanoma (MM) is a major problem today. In this study, we aimed to investigate the correlations of two inflammatory biomarkers-serum amyloid A (SAA) and lipoprotein associated phospholipase A 2 (Lp-PLA(2))-in clinical follow-up and staging of MM. Methods: Lactate dehydrogenase (LDH) and C-reactive protein (CRP) (Routine), SAA and S100B (ELISA), and Lp-PLA(2) (PLAC (R) Test) activity levels were examined in histologically and clinically confirmed MM patients (n=131) and in healthy controls (n=27). Results: Sedimentation rate and LDH, CRP, S100B, SAA, Lp-PLA(2) activities were found to be significantly higher in MM patients compared to control group (p<0.05). SAA showed the strongest correlation with disease stage (Spearman's correlation coefficient=0.622, p=0.000). Receiver operating characteristic analysis revealed that SAA exhibited the largest area under the curve=0.984, p=0.000, highest sensitivity (95%) and specificity (93%). Pearson's test indicated a weak positive correlation between SAA levels and Lp-PLA(2) activity (r=0.311, p=0.000). Conclusion: This is the first study to evaluate the activity of inflammatory biomarker Lp-PLA(2) in melanoma patients. Both SAA and Lp-PLA(2) were in highest levels in stage 4 patients, and they are thought to be candidate biomarkers to be used in detecting tumor progression. According to our results, SAA is the biomarker which correlates best with disease stage in MM.Ege University Research Project Fund [Tip-076]This work was funded by a fund from Ege University Research Project Fund (Tip-076)

Combining clinical features and MEST-C score in IgA nephropathy may be a better determinant of kidney survival

Introduction. IgA nephropathy (IgAN) is a heterogeneous disease with highly variable clinical and histopathological features. We investigated the effects of Oxford classification and clinical features on renal survival in patients with IgAN.Methods. This retrospective observational study conducted from 2013 to 2017. Ninety-seven patients who were followed up more than six months were examined.Results. A total of 97 patients (68% male and median age 40 years) were enrolled in this study. 13% of patients developed end stage renal disease (ESRD) within the median of 37 months of follow-up. Need for renal replacement therapy at the time of diagnosis, serum creatinine level of higher than 1.97 mg/dl, serum albumin level less than 3.5 gr/dl, 24-hour urine protein level of higher than > 3.5 g/day, the percentage of glomerulosclerosis higher than 53%, T2 score and total MEST-C score higher than two were found to be significant predictors of development of ESRD. None of the clinical or histopathological features were found to be significant predictor of steroid treatment sensitivity except T1-2 scores.Conclusion. We think that IgA nephropathy is a heterogeneous disease that requires clinical and histopathological features to be evaluated together, but not individually, to determine renal survival.What is new. Iga nephropathy is a heterogeneous disease and modern pathologic classification systems is not enough to predict to prognosis. Histopathological features to be evaluated with clinical features, but not individually, to determine renal survival. Also glucocorticoid treatment response seems to be independent from clinical and histopathological features except T1-2 score

Combining clinical features and MEST-C score in IgA nephropathy may be a better determinant of kidney survival

Introduction. IgA nephropathy (IgAN) is a heterogeneous disease with highly variable clinical and histopathological features. We investigated the effects of Oxford classification and clinical features on renal survival in patients with IgAN

Congenital Factor Deficiencies in Children: A Report of a Single-Center Experience

Congenital factor deficiencies (CFDs) refer to inherited deficiency of coagulation factors in the blood. A total of 481 patients with CFDs, who were diagnosed and followed at our Pediatric Hematology and Oncology Clinic between 1990 and 2015, were retrospectively evaluated. Of the 481 cases, 134 (27.8%) were hemophilia A, 38 (7.9%) were hemophilia B, 57 (11.8%) were von Willebrand disease (vWD), and 252 (52.3%) were rare bleeding disorders (RBDs). The median age of the patients at the time of diagnosis and at the time of the study was 4.1 years (range: 2 months to 20.4 years) and 13.4 years (range: 7 months to 31.3 years), respectively. The median duration of the follow-up time was 6.8 years (range: 2.5 months to 24.8 years). One hundred nineteen (47.2%) of 252 patients with RBDs were asymptomatic, 49 (41.1%) of whom diagnosed by family histories, 65 (54.6%) through preoperative laboratory studies, and 5 (4.2%) after prolonged bleeding during surgeries. Consanguinity rate for the RBDs was 47.2%. Prophylactic treatment was initiated in 80 patients, 58 of whom were hemophilia A, 7 were hemophilia B, 13 were RBDs, and 2 were vWD. Significant advances have been achieved during the past 2 decades in the treatment of patients with CFDs, particularly in patients with hemophilias. The rarity and clinical heterogeneity of RBDs lead to significant diagnostic challenges and improper management. In this regard, multinational collaborative efforts are needed with the hope that can improve the management of patients with RBDs

Discovery and characterization of orally bioavailable 4-chloro-6-fluoroisophthalamides as covalent PPARG inverse-agonists

PPAR gamma (PPARG) is a ligand activated transcription factor that regulates genes involved in inflammation, bone biology, lipid homeostasis, as well as a master regulator of adipogenesis and a potential lineage driver of luminal bladder cancer. While PPARG agonists lead to transcriptional activation of canonical target genes, inverse agonists have the opposite effect through inducing a transcriptionally repressive complex leading to repression of canonical target gene expression. While many agonists have been described and tested clinically, inverse agonists offer an underexplored avenue to modulate PPARG biology in vivo. Current inverse agonists lack favorable in vivo properties; herein we describe the discovery and characterization of a series of orally bioavailable 4-chloro-6-fluoroisophthalamides as covalent PPARG inverse-agonists, BAY-5516, BAY-5094, and BAY-9683. Structural studies of this series revealed distinct pre- and post-covalent binding positions, which led to the hypothesis that interactions in the pre-covalent conformation are primarily responsible for driving affinity, while interactions in the post-covalent conformation are more responsible for cellular functional effects by enhancing PPARG interactions with its corepressors. The need to simultaneously optimize for two distinct states may partially explain the steep SAR observed. Exquisite selectivity was achieved over related nuclear receptors in the subfamily due in part to a covalent warhead with low reactivity through an SNAr mechanism in addition to the specificity gained through covalent binding to a reactive cysteine uniquely positioned within the PPARG LBD. BAY-5516, BAY-5094, and BAY-9683 lead to pharmacodynamic regulation of PPARG target gene expression in vivo comparable to known inverse agonist SR10221 and represent new tools for future in vivo studies to explore their potential utility for treatment of disorders of hyperactivated PPARG including luminal bladder cancer and other disorders

Association of biochemical and clinical parameters with parathyroid adenoma weight. Turkish-Bulgarian endocrine and breast surgery study group, hyperparathyroidism registry study

Background: Primary hyperparathyroidism (pHPT) caused by a single benign parathyroid adenoma is a common endocrine disorder that is affected by regional differences. Living in different geographical regions reveals differences in the laboratory results and pathological findings, but studies on this subject are not sufficient. The article focuses on biochemical and pathological effects of geographical differences in parathyroid adenoma. In addition, the present study seeks to elaborate on treatment methods and effectiveness of screening in geographical area of Bulgaria and Turkey. Method: In this prospective study, 159 patients were included from 16 centres. Demographic characteristics, symptoms, biochemical markers and pathologic characteristics were analysed and compared between 8 different regions. Results: Patients from Turkish Black Sea had the highest median serum calcium (Ca) level, whereas patients from Eastern Turkey had the lowest median serum phosphorus (P) level. On the other hand, there was no significant difference between Ca, parathormone (PTH) and P levels according to regions. Patients from Eastern Turkey had the highest adenoma weight, while patients from Bulgaria had the lowest adenoma weight. The weight of adenoma showed statistically significant differences between regions (p < 0.001). There was a correlation between adenoma weight and serum PTH level (p = 0.05) and Ca level (p = 0.035). Conclusion: This study has provided a deeper insight into the effect of the regional differences upon clinicopathological changing and biochemical values of pHTP patients with adenoma. Awareness of regional differences will assist in biochemical screening and treatment of this patient group. (c) 2021 Asian Surgical Association and Taiwan Robotic Surgery Association. Publishing services by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/)