ZraP is a periplasmic molecular chaperone and a repressor of the zinc-responsive two-component regulator ZraSR

The bacterial envelope is the interface with the surrounding environment and is consequently subjected to a barrage of noxious agents including a range of compounds with antimicrobial activity. The ESR (envelope stress response) pathways of enteric bacteria are critical for maintenance of the envelope against these antimicrobial agents. In the present study, we demonstrate that the periplasmic protein ZraP contributes to envelope homoeostasis and assign both chaperone and regulatory function to ZraP from Salmonella Typhimurium. The ZraP chaperone mechanism is catalytic and independent of ATP; the chaperone activity is dependent on the presence of zinc, which is shown to be responsible for the stabilization of an oligomeric ZraP complex. Furthermore, ZraP can act to repress the two-component regulatory system ZraSR, which itself is responsive to zinc concentrations. Through structural homology, ZraP is a member of the bacterial CpxP family of periplasmic proteins, which also consists of CpxP and Spy. We demonstrate environmental co-expression of the CpxP family and identify an important role for these proteins in Salmonella's defence against the cationic antimicrobial peptide polymyxin B

Heterogeneity of viral IL-6 expression in HHV-8-associated diseases.

In order to characterize the expression of the viral interleukin-6 (vIL-6) homologue in various human herpesvirus 8 (HHV-8)-associated diseases, in situ hybridization and immunohistochemistry were applied to formalin-fixed specimens. These assays showed consistent expression of vIL-6 in primary effusion lymphomas and in a case of human immunodeficiency virus (HIV)-associated lymphadenopathy with a Castleman's disease-like appearance. In contrast, Kaposi's sarcoma specimens showed marked differences among specimens. In a consecutive series of specimens from the Johns Hopkins archives, vIL-6 expression was demonstrated in one of 13 cases. However, among 7 specimens selected from the AIDS Malignancy Bank because of their high levels of the T1.1 lytic transcript and virion production, vIL-6 expression was consistently demonstrated in infiltrating mononuclear cells and occasional spindle-shaped cells. Thus vIL-6 expression in clinical specimens correlates with other measures of the lytic viral cycle. Both assays generally give congruent results and are consistent with the possibility that vIL-6 expression plays a role in the pathogenesis of a variety of HHV-8-associated diseases

Experience with MRD Testing in B- ALL By Flow Cytometry Does Not Prevent Interpretative Discordance

Abstract Background: Minimal residual disease (MRD) in B lymphoblastic leukemia (B-ALL) as measured by flow cytometry is well-established as an important prognostic factor; Its presence is used to adjust treatment in most therapeutic protocols in children , while the lack of a standardized assay has hampered the introduction of flow cytometric MRD in adult ALL trials. On the other hand, measuring MRD has become part of the standard of care even for patients not on clinical trials. Although flow cytometric analysis of MRD in B-ALL has been well standardized in clinical trials of the Children's Oncology Group (COG) in North America (Borowitz et al Blood 2015;126:964), there are no data on performance characteristics of this assay within routine clinical labs. Methods: As part of an ongoing effort to standardize and decentralize ALL MRD measurement, list-mode data from post-induction marrows were distributed from one COG reference lab to 7 different clinical flow cytometry labs self-identified as having experience with ALL MRD. All labs were provided with the COG protocol used for MRD analysis along with a template illustrating recommended gating strategies, and formulas for calculating MRD burden. List-mode files of pre-treatment B-ALL samples analyzed with the standard COG B-ALL MRD antibody panel were distributed for comparison. In the first rounds, list-mode files from 15 samples were distributed to the 7 labs. Samples included those with and without MRD as assayed in the reference lab. Samples were selected to include normal B-cell precursors (hematogones) or MRD that had undergone phenotypic change with therapy. Some samples had artifacts that could potentially mimic small MRD populations. To improve performance, educational sessions were implemented, and 10 more list-mode file samples were distributed in a second round of challenges. Results: There was considerable dispersion of MRD results among the 7 labs that analyzed the list-mode files (Fig 1A). Although high levels of MRD were uniformly recognized, several labs misclassified normal B-cell precursors and/or mischaracterized small artifacts as MRD. Moreover, among samples correctly identified as positive, quantitative differences in MRD levels from those reported by the reference lab were seen. Among 95 total challenges, the overall discordance rate was 24%. This included 11 false positives, 7 false negatives, and an additional 5 quantitatively discordant cases among positives (defined as outside +/- 0.5 log of the reference lab value). In the second round, positive and negative samples, as well as those with normal precursors were included, though these samples contained fewer artifacts than those of the first round. Performance improved considerably (Fig 1B); out of 70 challenges, there were 5 false positives and 1 false negative (8.6% discordance), and no cases were quantitatively discordant. Four of the 6 deviations occurred in a single lab. Three samples with hematogones were still misclassified as MRD. Conclusions: Despite the provision of a standardized analysis protocol, even experienced laboratories have difficulty with B-ALL MRD analysis by flow cytometry. Some of these difficulties can be overcome with education, but even with education recognition of hematogones still remains a challenge for some labs. Extrapolating these results to other laboratories with less experience indicates the need for caution in migrating MRD testing from centralized reference laboratories, and suggests that implementation of MRD testing as part of routine clinical management of B-ALL patients in a manner similar to that of routine flow cytometric classification of leukemia may require additional training and resources. Figure 1 Figure 1. Disclosures Wood: Pfizer: Honoraria, Other: Laboratory Services Agreement; Juno: Other: Laboratory Services Agreement; Amgen: Honoraria, Other: Laboratory Services Agreement; Seattle Genetics: Honoraria, Other: Laboratory Services Agreement. Lozanski:Stemline Therapeutics Inc.: Research Funding; Beckman Coulter: Research Funding; Boehringer Ingelheim: Research Funding; Genentech: Research Funding. Mukundan:CCS Associates: Employment. Higley:CCS Associates: Employment. Sigman:CCS Associates: Equity Ownership. Borowitz:BD Biosciences: Research Funding; Medimmune: Research Funding; Bristol Myers Squibb: Research Funding; HTG Molecular: Consultancy. </jats:sec