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Background: The germline TP53-R337H mutation is strongly associated with pediatric adrenocortical tumors (ACT) in southern Brazil; it has low penetrance and limited tissue specificity in most families and therefore is not associated with Li-Fraumeni syndrome. However, other tumor types, mainly breast cancer, have been observed in carriers of several unrelated kindreds, raising the possibility that the R337H mutation may also contribute to breast tumorigenesis in a genetic background-specific context.Methods: We conducted a case-control study to determine the prevalence of the R337H mutation by sequencing TP53 exon 10 in 123 women with breast cancer and 223 age- and sex-matched control subjects from southern Brazil. Fisher's test was used to compare the prevalence of the R337H.Results: The R337H mutation was found in three patients but in none of the controls (p = 0.0442). Among the carriers, two had familial history of cancer meeting the Li-Fraumeni-like criteria. Remarkably, tumors in each of these three cases underwent loss of heterozygosity by eliminating the mutant TP53 allele rather than the wild-type allele. Polymorphisms were identified within the TP53 (R72P and Ins16) and MDM2 (SNP309) genes that may further diminish TP53 tumor suppressor activity.Conclusion: These results demonstrate that the R337H mutation can significantly increase the risk of breast cancer in carriers, which likely depends on additional cooperating genetic factors. These findings are also important for understanding how low-penetrant mutant TP53 alleles can differentially influence tumor susceptibility. [ABSTRACT FROM AUTHOR]

Yeast is an essential model organism for studying protein ubiquitination pathways; however, identifying the direct substrates of E3 in the cell presents a challenge. Here, we present a protocol for using the orthogonal ubiquitin transfer (OUT) cascade to profile the substrate specificity of yeast E3 Rsp5. We describe steps for OUT profiling, proteomics analysis, in vitro and in cell ubiquitination, and stability assay. The protocol can be adapted for identifying and verifying the ubiquitination targets of other E3s in yeast. For complete details on the use and execution of this protocol, please refer to Wang et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Attachment of the ubiquitin (UB) peptide to proteins via the E1-E2-E3 enzymatic machinery regulates diverse biological pathways, yet identification of the substrates of E3 UB ligases remains a challenge. We overcame this challenge by constructing an "orthogonal UB transfer" (OUT) cascade with yeast E3 Rsp5 to enable the exclusive delivery of an engineered UB (xUB) to Rsp5 and its substrate proteins. The OUT screen uncovered new Rsp5 substrates in yeast, such as Pal1 and Pal2, which are partners of endocytic protein Ede1, and chaperones Hsp70-Ssb, Hsp82, and Hsp104 that counteract protein misfolding and control self-perpetuating amyloid aggregates (prions), resembling those involved in human amyloid diseases. We showed that prion formation and effect of Hsp104 on prion propagation are modulated by Rsp5. Overall, our work demonstrates the capacity of OUT to deconvolute the complex E3-substrate relationships in crucial biological processes such as endocytosis and protein assembly disorders through protein ubiquitination., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Nasal osteotomy is a commonly performed procedure during rhinoplasty for both functional and cosmetic reasons. Teaching and learning this procedure proves difficult due to the reliance on nuanced tactile feedback. For surgical simulation, trainees are traditionally limited to cadaveric bones, which can be costly and difficult to obtain., Objective: This study aimed to design and print a low-cost midface model for nasal osteotomy simulation., Methods: A 3D reconstruction of the midface was modified using the free open-source design software Meshmixer (Autodesk Inc). The pyriform aperture was smoothed, and support rods were added to hold the fragments generated from the simulation in place. Several models with various infill densities were printed using a desktop 3D printer to determine which model best mimicked human facial bone., Results: A midface simulation set was designed using a desktop 3D printer, polylactic acid filament, and easily accessible tools. A nasal osteotomy procedure was successfully simulated using the model., Conclusions: 3D printing is a low-cost, accessible technology that can be used to create simulation models. With growing restrictions on trainee duty hours, the simulation set can be used by programs to augment surgical training., (©Michelle Ho, Jared Goldfarb, Roxana Moayer, Uche Nwagu, Rohan Ganti, Howard Krein, Ryan Heffelfinger, Morgan Leigh Hutchinson. Originally published in JMIR Medical Education (http://mededu.jmir.org), 17.11.2020.)

Since September 2015, the World Health Organization has recommended antiretroviral therapy (ART) for all persons with human immunodeficiency virus (HIV) infection, regardless of clinical stage or CD4 count (1). This Treat All policy was based on evidence that ART initiation early in HIV infection as opposed to waiting for the CD4 count to decline to certain levels (e.g., <500 cells/mm 3 , per previous guidelines), was associated with reduced morbidity, mortality, and HIV transmission (2-4). Further, approximately half of persons enrolled in non-ART care that included monitoring for HIV disease progression (i.e., in pre-ART care) were lost to follow-up before becoming ART-eligible (5). India, the country with the third largest number of persons with HIV infection in the world (2.1 million), adopted the Treat All policy on April 28, 2017. This report describes implementation of Treat All during May 2017-June 2018, by India's National AIDS Control Organization (NACO) and partners, by facilitating ART initiation among persons previously in pre-ART care at 46 ART centers supported by the U.S. President's Emergency Plan for AIDS Relief (PEPFAR)* in six districts in the states of Maharashtra and Andhra Pradesh. Partners supported these 46 ART centers in identifying and attempting to contact persons who were enrolled in pre-ART care during January 2014-April 2017, and educating those reached about Treat All. ART center-based records were used to monitor implementation indicators, including ART initiation. A total of 9,898 (39.6%) of 25,007 persons previously enrolled in pre-ART care initiated ART; among these 9,898 persons, 6,315 (63.8%) initiated ART after being reached during May 2017-June 2018, including 1,635 (16.5%) who had been lost to follow-up before ART initiation. NACO scaled up efforts nationwide to build ART centers' capacity to implement Treat All. Active tracking and tracing of persons with HIV infection enrolled in care but not on ART, combined with education about the benefits of early HIV treatment, can facilitate ART initiation., Competing Interests: All authors have completed and submitted the ICMJE form for disclosure of potential conflicts of interest. No potential conflicts of interest were disclosed.

Rationale and Objectives: The purposes of this study were to provide a case-based overview of various immune-mediated side effects detected by 18F-Fluorodeoxyglucose (F-18 FDG) positron emission tomography-computed tomography (PET-CT) in the patients receiving ipilimumab immunotherapy for treatment of malignant melanoma, and discuss the importance of recognizing immune-mediated side effects in the use of F-18 FDG PET-CT for monitoring therapeutic effects of ipilimumab on metastatic melanoma., Materials and Methods: This is a retrospective case series study of the patients diagnosed with melanoma who were subjected to immunomodulating therapy with ipilimumab. F-18 FDG PET-CT findings were reviewed, and the patients with immune-mediated side effects were selected for further analysis, in conjunction with review of clinical progress notes, the results of laboratory tests, and findings of other imaging tests., Results: Four patients with immune-mediated side effects were identified among the patients being treated with ipilimumab and subjected to F-18 FDG PET-CT for monitoring therapeutic effects. These immune mediated side effects include new findings of abnormal increased FDG uptake associated with immune-mediated pancreatitis and hypophysitis, as well as immune-mediated thyroiditis and colitis reported previously., Conclusions: Various immune-mediated side effects were detected by F-18 FDG PET-CT in the patients subjected to immunomodulating therapy with ipilimumab. It is essential for the interpreting provider to recognize and differentiate abnormal FDG uptake associated with immune-mediated side effects from hypermetabolic malignant lesions when using F-18 FDG PET-CT for monitoring therapeutic effects of ipilimumab on melanoma lesions., (Copyright © 2017 The Association of University Radiologists. Published by Elsevier Inc. All rights reserved.)

The aggresome is an organelle that recruits aggregated proteins for storage and degradation. We performed an siRNA screen for proteins involved in aggresome formation and identified novel mammalian AAA+ protein disaggregases RuvbL1 and RuvbL2. Depletion of RuvbL1 or RuvbL2 suppressed aggresome formation and caused buildup of multiple cytoplasmic aggregates. Similarly, downregulation of RuvbL orthologs in yeast suppressed the formation of an aggresome-like body and enhanced the aggregate toxicity. In contrast, their overproduction enhanced the resistance to proteotoxic stress independently of chaperone Hsp104. Mammalian RuvbL associated with the aggresome, and the aggresome substrate synphilin-1 interacted directly with the RuvbL1 barrel-like structure near the opening of the central channel. Importantly, polypeptides with unfolded structures and amyloid fibrils stimulated the ATPase activity of RuvbL. Finally, disassembly of protein aggregates was promoted by RuvbL. These data indicate that RuvbL complexes serve as chaperones in protein disaggregation., (© 2015 The Authors.)

Quantitative phase imaging (QPI) utilizes the fact that the phase of an imaging field is much more sensitive than its amplitude. As fields from the source interact with the specimen, local variations in the phase front are produced, which provide structural information about the sample and can be used to reconstruct its topography with nanometer accuracy. QPI techniques do not require staining or coating of the specimen and are therefore nondestructive. Diffraction phase microscopy (DPM) combines many of the best attributes of current QPI methods; its compact configuration uses a common-path off-axis geometry which realizes the benefits of both low noise and single-shot imaging. This unique collection of features enables the DPM system to monitor, at the nanoscale, a wide variety of phenomena in their natural environments. Over the past decade, QPI techniques have become ubiquitous in biological studies and a recent effort has been made to extend QPI to materials science applications. We briefly review several recent studies which include real-time monitoring of wet etching, photochemical etching, surface wetting and evaporation, dissolution of biodegradable electronic materials, and the expansion and deformation of thin-films. We also discuss recent advances in semiconductor wafer defect detection using QPI.

To define the mutation spectrum in non-Down syndrome acute megakaryoblastic leukemia (non-DS-AMKL), we performed transcriptome sequencing on diagnostic blasts from 14 pediatric patients and validated our findings in a recurrency/validation cohort consisting of 34 pediatric and 28 adult AMKL samples. Our analysis identified a cryptic chromosome 16 inversion (inv(16)(p13.3q24.3)) in 27% of pediatric cases, which encodes a CBFA2T3-GLIS2 fusion protein. Expression of CBFA2T3-GLIS2 in Drosophila and murine hematopoietic cells induced bone morphogenic protein (BMP) signaling and resulted in a marked increase in the self-renewal capacity of hematopoietic progenitors. These data suggest that expression of CBFA2T3-GLIS2 directly contributes to leukemogenesis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Pediatric de novo acute myeloid leukemia (AML) is an aggressive malignancy with current therapy resulting in cure rates of only 60%. To better understand the cause of the marked heterogeneity in therapeutic response and to identify new prognostic markers and therapeutic targets a comprehensive list of the genetic mutations that underlie the pathogenesis of AML is needed. To approach this goal, we examined diagnostic leukemic samples from a cohort of 111 children with de novo AML using single-nucleotide-polymorphism microarrays and candidate gene resequencing. Our data demonstrate that, in contrast to pediatric acute lymphoblastic leukemia (ALL), de novo AML is characterized by a very low burden of genomic alterations, with a mean of only 2.38 somatic copy-number alterations per leukemia, and less than 1 nonsynonymous point mutation per leukemia in the 25 genes analyzed. Even more surprising was the observation that 34% of the leukemias lacked any identifiable copy-number alterations, and 28% of the leukemias with recurrent translocations lacked any identifiable sequence or numerical abnormalities. The only exception to the presence of few mutations was acute megakaryocytic leukemias, with the majority of these leukemias being characterized by a high number of copy-number alterations but rare point mutations. Despite the low overall number of lesions across the patient cohort, novel recurring regions of genetic alteration were identified that harbor known, and potential new cancer genes. These data reflect a remarkably low burden of genomic alterations within pediatric de novo AML, which is in stark contrast to most other human malignancies.

Purpose: In proliferative vitreoretinopathy (PVR), retinal pigment epithelial (RPE) cells enter the vitreous and proliferate. They become fibroblast-like and participate in the formation of contractile membranes, which can lead to retinal detachment. Vitreous treatment of RPE cells in vitro results in similar morphologic changes. This study was conducted to examine vitreous-induced modulation of gene expression in RPE cells., Methods: Low-passage human RPE cell lines derived from three donors were each treated for 6, 12, 24, or 48 hours with complete medium or complete medium containing 25% vitreous. Changes in mRNA levels were examined by using microarrays. Real-time quantitative PCR (qPCR) was used to measure mRNA expression of a subset of genes in cells from three additional donors. Immunohistochemistry and immunoblot analysis were used to examine protein expression., Results: Vitreous treatment caused a progressive reprogramming of gene expression. qPCR confirmed vitreous modulation of mRNA levels of 10 of 10 genes. Changes consistent with a transition from an epithelial to a mesenchymal phenotype were observed. Downregulated genes included genes associated with differentiated RPE cells. Upregulated genes included genes associated with stress and inflammation. Pathway analysis indicated that the transforming growth factor-beta/bone morphogenetic protein (BMP) pathway and the focal adhesion pathway may play a role in this process. BMP-2 protein and mRNA were increased., Conclusions: Despite the biological variation in vitreous and RPE donors, vitreous reproducibly modulated a limited number of mRNAs. Many of these changes were consistent with the more fibroblast-like appearance of vitreous-treated cells and with the pathobiology of PVR. TGF-beta and BMP-2 may be important modulators of vitreous-induced changes in gene expression.

Purpose: When human retinal pigment epithelial (RPE) cells come in contact with vitreous, they undergo changes in gene expression that include inflammatory and anti-oxidant responses. The effects of vitreous on expression of heme oxygenase-1 (HO-1), metallothionein (MT) -1a and -2a, and c-fos were investigated. Activator protein-1 (AP-1) binding sites are located in the promoter region of HO-1 and MT genes and the effects of vitreous on c-fos activity were investigated., Methods: Low passage cultures of human RPE cells were grown in the presence or absence of vitreous or transforming growth factor-beta (TGF-beta). The expression of HO-1 and MTs was measured by real time PCR and, in the case of HO-1, by immunoblotting and immunofluorescence microscopy. Specific inhibitors were used to investigate possible signaling pathways. The effect of vitreous on activation of AP-1 transcription factor was determined by immunoblotting, electrophoretic mobility shift assays, or immunofluorescence microscopy., Results: Incubation of RPE cells with vitreous resulted in increased expression of HO-1, MT-1a and MT-2a. TGF-beta caused an increase in HO-1 expression, although not to the extent mediated by vitreous, but had little effect on MT expression. Addition of inhibitors of TGF-beta signaling (SB431542 or TGF-beta-neutralizing antibodies) decreased the vitreous induction of HO-1. Several reactive oxygen species (ROS) quenchers inhibited the TGF-beta-induced or vitreous-induced elevation of HO-1 mRNA but had no effect on vitreous-mediated induction of MT expression. Inhibitors of the mitogen-activated protein kinase (p38MAPK; SB203580) and Jun N-terminal kinase (JNK; SP600125) pathways inhibited vitreous-induction of HO-1. C-fos, a component of AP-1 transcription factor complexes, exhibited increased expression and activation in the presence of vitreous., Conclusions: TGF-beta, a known component of vitreous, can account for some but not all of the regulation of the anti-oxidant, anti-inflammatory HO-1 gene in human RPE cells, but it does not participate in the vitreous-mediated upregulation of MTs. Both vitreous and TGF-beta signals increased HO-1 expression via ROS but the latter were not involved in vitreous-mediated MT expression. Increased p38, JNK, and c-fos activation may be implicated in vitreous modulation of HO-1.

Both epidermal growth factor receptor (EGFR) and ErbB-2 play an important role in cancer biology and constitute promising molecular targets of therapy. EGFR and ErbB-2 expression has been observed in rhabdomyosarcoma cell lines but not analyzed systematically in rhabdomyosarcoma tumors. Tissue microarray sections representing 66 rhabdomyosarcoma tumors (34 embryonal rhabdomyosarcoma, 32 alveolar rhabdomyosarcoma) were surveyed by immunohistochemistry using antibodies specific for EGFR and ErbB-2. Immunostains were assessed for intensity (0: no immunostaining; 1: weak; 2: moderate; 3: strong) and percentage of at least 500 neoplastic cells exhibiting membranous or membranous and cytoplasmic immunostaining. EGFR and ErbB-2 expression was considered positive if the product of intensity and percentage was greater than 10. Patients had a median age of 5.7 years (range 8 months-19.1 years), and of 65/66 patients, 38 were males and 27 were females. Expression of ErbB-2 was identified in 22/66 (33%) cases and tended to be more frequent in the alveolar subtype (13/32, 41%, vs 9/34, 26%, P=0.30). Expression of EGFR was identified in 31/66 (47%) cases and correlated with the embryonal subtype (26/34, 76%, vs 5/32, 16%, P<0.0001) independent of stage, age, and gender. Coexpression of EGFR and ErbB-2 was identified in eight tumors, of which six were embryonal rhabdomyosarcoma. None of the cases exhibited EGFR or ErbB-2 gene amplification, as assessed using fluorescence in situ hybridization. Furthermore, analysis of 11 additional rhabdomyosarcoma tumors (six alveolar; five embryonal) revealed no evidence of mutations in EGFR exons 18, 19, 20, and 21. In summary, expression of EGFR and/or ErbB-2 is detected in a sizeable subset of rhabdomyosarcoma tumors without evidence of EGFR or ErbB-2 amplification or mutations in the EGFR tyrosine kinase domain. Notably, expression of EGFR correlates with the embryonal subtype, which is also more likely to coexpress EGFR and ErbB-2., (Published online 26 May 2006.)

Purpose: To investigate the alterations in gene expression when human retinal pigment epithelial (RPE) cells in culture are treated with vitreous as a model for the changes that occur in proliferative vitreoretinopathy., Methods: Human RPE cells were cultured with or without human vitreous or collagen. RNA was extracted and reverse transcribed. The RNAs expressed were compared by using DNA macroarrays. Messenger RNA levels were also measured using real-time reverse transcription polymerase chain reaction. Protein expression was examined by immunoblot analysis. Immunoassays were used to determine levels of prostaglandin E(2)., Results: Vitreous treatment of RPE cells resulted in increased expression of two critical enzymes in the synthesis of prostaglandin E(2): membrane-associated prostaglandin E-synthase (mPGES) and cyclooxygenase (COX)-2. Increased levels of mPGES RNA and protein were still present at 48 hours of treatment, but the increase in COX-2 mRNA and protein was transient. The increase in the expression of mPGES was associated with an increase in the production of prostaglandin E(2) that was observed at 12 and 24 hours of treatment but not at 48 hours. Treatment with 100 microg collagen I per ml medium did not cause increased expression of mPGES and COX-2, even though both collagen- and vitreous-treatment caused a morphologic change in the RPE cells to a more fibroblast-like phenotype., Conclusions: Treatment of human RPE cells with vitreous induces changes in gene expression that are indicative of an inflammatory response.