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3-Bromopyruvate (3BP) is a potential anticancer agent viewed as a glycolytic inhibitor that preferentially kills cancer cells through inhibition of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), resulting in severe energy depletion. We previously identified four cysteine residues in GAPDH that are alkylated by 3BP, resulting in its inactivation. However, we also showed that addition of excess pyruvate, the final product of glycolysis, was unable to rescue cells from 3BP treatment. This result indicates that GAPDH may not be the only relevant target and is consistent with the chemical reactivity of 3BP that should result in the modification of cysteine residues in many different proteins. To directly test this hypothesis, we first synthesized a probe of 3BP activity bearing an alkyne functionality, termed AO3BP, and then demonstrated that this probe could modify a variety of proteins in living cells. Subsequent competition of AO3BP labeling with pretreatment by 3BP identified 62 statistically significant proteins of various functions as targets of 3BP, confirming that 3BP labeling is indeed widespread. We conclude that 3BP's cytotoxic impact on cancer cells does not only result from selective inhibition of glycolysis but rather from a more widespread effect on cellular proteins that could be driven by the pharmacokinetics of the 3BP. These pleiotropic consequences should be considered when thinking about the potential toxicity of this highly reactive compound.

Objective Deficiency in the cytosolic DNA sensor RNA Polymerase III (POL III) was recently described in children with severe varicella-zoster virus (VZV) infection in the CNS or lungs. Here, we describe a pair of monozygotic female twins, who both experienced severe recurrent CNS vasculitis caused by VZV reactivation. The clinical presentation and findings included recurrent episodes of headache, dizziness, and neurologic deficits, CSF with pleocytosis and intrathecal VZV antibody production, and MRI of the brain showing ischemic lesions. Methods We performed whole-exome sequencing and identified a rare mutation in the POL III subunit POLR3F. Subsequently, antiviral responses in patient peripheral blood mononuclear cells (PBMCs) were examined and compared with healthy controls. Results The identified R50W POLR3F mutation is predicted by bioinformatics to be damaging, and when tested in functional assays, patient PBMCs exhibited impaired antiviral and inflammatory responses to the POL III agonist poly(dA:dT) and increased viral replication compared with controls. Conclusions Altogether, these cases add genetic and immunologic evidence to the novel association between defects in sensing of AT-rich DNA present in the VZV genome and increased susceptibility to severe manifestations of VZV infection in the CNS in humans.

The N-glycans attached to the Fab and Fc domains play distinct roles in modulating the functions of antibodies. However, post-translational site-selective modifications of glycans in antibodies and other multiply glycosylated proteins remain a challenging task. Here, we report a chemoenzymatic method that permits independent manipulation of the Fab and Fc N-glycans, using cetuximab as a model therapeutic monoclonal antibody. Taking advantage of the substrate specificity of three endoglycosidases (Endo-S, Endo-S2, and Endo-F3) and their glycosynthase mutants, together with an unexpected substrate site-selectivity of a bacterial alpha 1,6-fucosidase from Lactobacillus casei (AlfC), we were able to synthesize an optimal homogeneous glycoform of cetuximab in which the heterogeneous and immunogenic Fab N-glycans were replaced with a single sialylated N-glycan, and the core-fucosylated Fc N-glycans were remodeled with a nonfucosylated and fully galactosylated N-glycan. The glycoengineered cetuximab demonstrated increased affinity for the Fc gamma IIIa receptor and significantly enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity.

Mature microRNAs (miRNAs) are a class of small noncoding RNA molecules involved in regulation of post-translational gene expression. Although aberrant levels of miRNAs have been found in various tumor tissues, their importance in tumor development and the molecular basis of their regulatory role remain unclear. Our bioinformatic analysis on The Cancer Genome Atlas database and microarray-based comparison of miRNA in different cell lines revealed that the level of mir-1287 is suppressed in hepatocellular carcinoma (HCC) cells. When upregulated, mir-1287 can reduce the tumorigenesis phenotypes of HCC cells in several in vitro models. We further found that mir-1287 directly targets messenger RNA encoding PIK3R3, which is a tumor-promoting factor acting in several pathways linked to tumorigenesis. Our study suggests that aberrant suppression of mir-1287 is potentially responsible for the development of HCC, and miRNA-based strategies may be developed for efficient detection and treatment of HCC.

Piezo1 is an ion channel that gates open when mechanical force is applied to a cell membrane, thus allowing cells to detect and respond to mechanical stimulation. Molecular structures of Piezo1 reveal a large ion channel with an unusually curved shape. This study analyzes how such a curved ion channel interacts energetically with the cell membrane. Through membrane mechanical calculations, we show that Piezo1 deforms the membrane shape outside the perimeter of the channel into a curved 'membrane footprint'. This membrane footprint amplifies the sensitivity of Piezo1 to changes in membrane tension, rendering it exquisitely responsive. We assert that the shape of the Piezo channel is an elegant example of molecular form evolved to optimize a specific function, in this case tension sensitivity. Furthermore, the predicted influence of the membrane footprint on Piezo gating is consistent with the demonstrated importance of membrane-cytoskeletal attachments to Piezo gating.

As members of the National Academies of Sciences, Engineering, and Medicine committee that wrote the report on the return of individual research results (1), we reject the allegations in the Policy Forum “Return of results and data to study participants” (S. M. Wolf and B. J. Evans, 12 October, p. 159) that the report is paternalistic, misunderstands the law, burdens Institutional Review Boards (IRBs), and creates barriers to the return of results.

Background: The prevalence of nosocomial meticillin-resistant Staphylococcus aureus (MRSA) was previously estimated as 23% in a paediatric hospital in Luanda, Angola and 18% in a general hospital in Sao Tome and Principe. Aim: To evaluate the prevalence of S. aureus/MRSA colonization among hospitalized children and their parents at two hospitals in Angola and Sao Tome and Principe. Methods: In 2017, 127 hospitalized children and 129 of their parents had nasal swabs for S. aureus/MRSA carriage in the two countries. The isolates were tested for the presence of the mecA and Panton-Valentine leukocidin (PVL) genes, and characterized by pulsed-field gel electrophoresis (PFGE), spa typing, multi-locus sequence typing and SCCmec typing. Findings: Twenty of 127 children (15.7%) and 13 of 129 parents (10.1%) were MRSA nasal carriers. Three lineages comprised 88% of the MRSA isolates: (i) PFGE A-ST5-SCCmec IVa (N=15; 45%), associated with spa type t105, recovered in Angola alone; (ii) PFGE N-ST8-IV/ V (N=7; 21%), associated with spa types t008/t121, recovered in Sao Tome and Principe alone; and (iii) PFGE B-ST88-IVa (N=7; 21%), associated with spa types t325/t786, present in both countries. Fifteen child/guardian pairs were colonized with identical MRSA (N=8) or meticillin-susceptible S. aureus (N=7) strains. PVL was detected in 25% of isolates, including two MRSA (ST30-V and ST8-IVa). Conclusion: Hospitalized children and their parents are important reservoirs of MRSA. Infection control measures should focus on parents in order to minimize the spread of MRSA to the community. (C) 2018 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Tumors often co-exist with T cells that recognize somatically mutated peptides presented by cancer cells on major histocompatibility complex I (MHC-I). However, it is unknown why the immune system fails to eliminate immune-recognizable neoplasms before they manifest as frank disease. To understand the determinants of MHC-I peptide immunogenicity in nascent tumors, we tested the ability of thousands of MHC-I ligands to cause tumor subclone rejection in immunocompetent mice by use of a new 'PresentER' antigen presentation platform. Surprisingly, we show that immunogenic tumor antigens do not lead to immune-mediated cell rejection when the fraction of cells bearing each antigen ('clonal fraction') is low. Moreover, the clonal fraction necessary to lead to rejection of immunogenic tumor subclones depends on the antigen. These data indicate that tumor neoantigen heterogeneity has an underappreciated impact on immune elimination of cancer cells and has implications for the design of immunotherapeutics such as cancer vaccines.