A Quantile-Quantile Toolbox for Reference Intervals.

BACKGROUND: Parametric statistical methods are generally better than nonparametric, but require that data follow a known, usually normal, distribution. One important application is finding reference limits and detection limits. Parametric analyses yield better estimates and measures of their uncertainty than nonparametric approaches, which rely solely on a few extreme values. Some reference data follow normal distributions; some can be transformed to normal; some are normal or transformable to normal apart from a few extreme values; and detection and quantitation limits can lead to data censoring. METHODS: A quantile-quantile (QQ) toolbox provides powerful general methodology for all these settings. RESULTS: QQ methodology leads to a family of simple methods for finding optimal power transformations, testing for normality before and after transformation, estimating reference limits, and constructing confidence intervals. CONCLUSIONS: These parametric methods have a particular appeal to clinical laboratorians because, while statistically rigorous, they do not require specialized software or statistical expertise, but can be implemented even in spreadsheets. We conclude with an exploration of reference values for amyloid beta proteins associated with Alzheimer disease.

The Alzheimer's Association international guidelines for handling of cerebrospinal fluid for routine clinical measurements of amyloid ß and tau.

The core cerebrospinal fluid (CSF) Alzheimer's disease (AD) biomarkers amyloid beta (Aß42 and Aß40), total tau, and phosphorylated tau, have been extensively clinically validated, with very high diagnostic performance for AD, including the early phases of the disease. However, between-center differences in pre-analytical procedures may contribute to variability in measurements across laboratories. To resolve this issue, a workgroup was led by the Alzheimer's Association with experts from both academia and industry. The aim of the group was to develop a simplified and standardized pre-analytical protocol for CSF collection and handling before analysis for routine clinical use, and ultimately to ensure high diagnostic performance and minimize patient misclassification rates. Widespread application of the protocol would help minimize variability in measurements, which would facilitate the implementation of unified cut-off levels across laboratories, and foster the use of CSF biomarkers in AD diagnostics for the benefit of the patients.

Protection against Borreliella burgdorferi infection mediated by a synthetically engineered DNA vaccine.

Lyme disease is the most common vector-borne disease in North America. The etiological agent is the spirochete Borreliella burgdorferi, transmitted to mammalian hosts by the Ixodes tick. In recent years there has been an increase in the number of cases of Lyme disease. Currently, there is no vaccine on the market for human use. We describe the development of a novel synthetically engineered DNA vaccine, pLD1 targeting the outer-surface protein A (OspA) of Borreliella burgdorferi. Immunization of C3 H/HeN mice with pLD1 elicits robust humoral and cellular immune responses that confer complete protection against a live Borreliella burgdorferi bacterial challenge. We also assessed intradermal (ID) delivery of pLD1 in Hartley guinea pigs, demonstrating the induction of robust and durable humoral immunity that lasts at least 1 year. We provide evidence of the potency of pLD1 by showing that antibodies targeting the OspA epitopes which have been associated with protection are prominently raised in the immunized guinea pigs. The described study provides the basis for the advancement of pDL1 as a potential vaccine for Lyme disease control.

In Vivo Delivery of a DNA-Encoded Monoclonal Antibody Protects Non-human Primates against Zika Virus.

Zika virus (ZIKV) infection is endemic to several world regions, and many others are at high risk for seasonal outbreaks. Synthetic DNA-encoded monoclonal antibody (DMAb) is an approach that enables in vivo delivery of highly potent mAbs to control infections. We engineered DMAb-ZK190, encoding the mAb ZK190 neutralizing antibody, which targets the ZIKV E protein DIII domain. In vivo-delivered DMAb-ZK190 achieved expression levels persisting >10 weeks in mice and >3 weeks in non-human primate (NHPs), which is protective against ZIKV infectious challenge. This study is the first demonstration of infectious disease control in NHPs following in vivo delivery of a nucleic acid-encoded antibody, supporting the importance of this new platform.

Screening of a Haloferax volcanii Transposon Library Reveals Novel Motility and Adhesion Mutants.

Archaea, like bacteria, use type IV pili to facilitate surface adhesion. Moreover, archaeal flagella-structures required for motility-share a common ancestry with type IV pili. While the characterization of archaeal homologs of bacterial type IV pilus biosynthesis components has revealed important aspects of flagellum and pilus biosynthesis and the mechanisms regulating motility and adhesion in archaea, many questions remain. Therefore, we screened a Haloferax volcanii transposon insertion library for motility mutants using motility plates and adhesion mutants, using an adapted air-liquid interface assay. Here, we identify 20 genes, previously unknown to affect motility or adhesion. These genes include potential novel regulatory genes that will help to unravel the mechanisms underpinning these processes. Both screens also identified distinct insertions within the genomic region lying between two chemotaxis genes, suggesting that chemotaxis not only plays a role in archaeal motility, but also in adhesion. Studying these genes, as well as hypothetical genes hvo_2512 and hvo_2876-also critical for both motility and adhesion-will likely elucidate how these two systems interact. Furthermore, this study underscores the usefulness of the transposon library to screen other archaeal cellular processes for specific phenotypic defects.

Identification of Haloferax volcanii Pilin N-Glycans with Diverse Roles in Pilus Biosynthesis, Adhesion, and Microcolony Formation.

N-Glycosylation is a post-translational modification common to all three domains of life. In many archaea, the oligosacharyltransferase (AglB)-dependent N-glycosylation of flagellins is required for flagella assembly. However, whether N-glycosylation is required for the assembly and/or function of the structurally related archaeal type IV pili is unknown. Here, we show that of six Haloferax volcanii adhesion pilins, PilA1 and PilA2, the most abundant pilins in pili of wild-type and ΔaglB strains, are modified under planktonic conditions in an AglB-dependent manner by the same pentasaccharide detected on H. volcanii flagellins. However, unlike wild-type cells, which have surfaces decorated with discrete pili and form a dispersed layer of cells on a plastic surface, ΔaglB cells have thick pili bundles and form microcolonies. Moreover, expressing PilA1, PilA2, or PilA6 in ΔpilA[1-6]ΔaglB stimulates microcolony formation compared with their expression in ΔpilA[1-6]. Conversely, expressing PilA3 or PilA4 in ΔpilA[1-6] cells results in strong surface adhesion, but not microcolony formation, and neither pilin stimulates surface adhesion in ΔpilA[1-6]ΔaglB cells. Although PilA4 assembles into pili in the ΔpilA[1-6]ΔaglB cells, these pili are, unlike wild-type pili, curled, perhaps rendering them non-functional. To our knowledge, this is the first demonstration of a differential effect of glycosylation on pilus assembly and function of paralogous pilins. The growth of wild-type cells in low salt media, a condition that decreases AglB glycosylation, also stimulates microcolony formation and inhibits motility, supporting our hypothesis that N-glycosylation plays an important role in regulating the transition between planktonic to sessile cell states as a response to stress.

Archaeal type IV pili and their involvement in biofilm formation.

Type IV pili are ancient proteinaceous structures present on the cell surface of species in nearly all bacterial and archaeal phyla. These filaments, which are required for a diverse array of important cellular processes, are assembled employing a conserved set of core components. While type IV pilins, the structural subunits of pili, share little sequence homology, their signal peptides are structurally conserved allowing for in silico prediction. Recently, in vivo studies in model archaea representing the euryarchaeal and crenarchaeal kingdoms confirmed that several of these pilins are incorporated into type IV adhesion pili. In addition to facilitating surface adhesion, these in vivo studies also showed that several predicted pilins are required for additional functions that are critical to biofilm formation. Examples include the subunits of Sulfolobus acidocaldarius Ups pili, which are induced by exposure to UV light and promote cell aggregation and conjugation, and a subset of the Haloferax volcanii adhesion pilins, which play a critical role in microcolony formation while other pilins inhibit this process. The recent discovery of novel pilin functions such as the ability of haloarchaeal adhesion pilins to regulate swimming motility may point to novel regulatory pathways conserved across prokaryotic domains. In this review, we will discuss recent advances in our understanding of the functional roles played by archaeal type IV adhesion pili and their subunits, with particular emphasis on their involvement in biofilm formation.

A conserved type IV pilin signal peptide H-domain is critical for the post-translational regulation of flagella-dependent motility.

In many bacteria and archaea, type IV pili facilitate surface adhesion, the initial step in biofilm formation. Haloferax volcanii has a specific set of adhesion pilins (PilA1-A6) that, although diverse, contain an absolutely conserved signal peptide hydrophobic (H) domain. Data presented here demonstrate that these pilins (PilA1-A6) also play an important role in regulating flagella-dependent motility, which allows cells to rapidly transition between planktonic and sessile states. Cells lacking adhesion pilins exhibit a severe motility defect, however, expression of any one of the adhesion pilins in trans can rescue the motility and adhesion. Conversely, while deleting pilB3-C3, genes required for PilA pilus biosynthesis, results in cells lacking pili and having an adhesion defect, it does not affect motility, indicating that motility regulation requires the presence of pilins, but not assembled pili. Mutagenesis studies revealed that the pilin-dependent motility regulatory mechanism does not require the diverse C-terminal region of the PilA pilins but specifically involves the conserved H-domain. This novel post-translational regulatory mechanism, which employs components that promote biofilm formation to inhibit motility, can provide a rapid response to changing environmental conditions. A model for this regulatory mechanism, which may also be present in other prokaryotes, is discussed.

Haloferax volcanii cells lacking the flagellin FlgA2 are hypermotile.

Motility driven by rotational movement of flagella allows bacteria and archaea to seek favourable conditions and escape toxic ones. However, archaeal flagella share structural similarities with bacterial type IV pili rather than bacterial flagella. The Haloferax volcanii genome contains two flagellin genes, flgA1 and flgA2. While FlgA1 has been shown to be a major flagellin, the function of FlgA2 is elusive. In this study, it was determined that although FlgA2 by itself does not confer motility to non-motile ΔflgA1 Hfx. volcanii, a subset of these mutant cells contains a flagellum. Consistent with FlgA2 being assembled into functional flagella, FlgA1 expressed from a plasmid can only complement a ΔflgA1 strain when co-expressed with chromosomal or plasmid-encoded FlgA2. Surprisingly, a mutant strain lacking FlgA2, but expressing chromosomally encoded FlgA1, is hypermotile, a phenotype that is accompanied by an increased number of flagella per cell, as well as an increased flagellum length. Site-directed mutagenesis resulting in early translational termination of flgA2 suggests that the hypermotility of the ΔflgA2 strain is not due to transcriptional regulation. This, and the fact that plasmid-encoded FlgA2 expression in a ΔflgA2 strain does not reduce its hypermotility, suggests a possible regulatory role for FlgA2 that depends on the relative abundance of FlgA1. Taken together, our results indicate that FlgA2 plays both structural and regulatory roles in Hfx. volcanii flagella-dependent motility. Future studies will build upon the data presented here to elucidate the significance of the hypermotility of this ΔflgA2 mutant, and will illuminate the regulation and function of archaeal flagella.

Novel archaeal adhesion pilins with a conserved N terminus.

Type IV pili play important roles in a wide array of processes, including surface adhesion and twitching motility. Although archaeal genomes encode a diverse set of type IV pilus subunits, the functions for most remain unknown. We have now characterized six Haloferax volcanii pilins, PilA[1-6], each containing an identical 30-amino-acid N-terminal hydrophobic motif that is part of a larger highly conserved domain of unknown function (Duf1628). Deletion mutants lacking up to five of the six pilin genes display no significant adhesion defects; however, H. volcanii lacking all six pilins (ΔpilA[1-6]) does not adhere to glass or plastic. Consistent with these results, the expression of any one of these pilins in trans is sufficient to produce functional pili in the ΔpilA[1-6] strain. PilA1His and PilA2His only partially rescue this phenotype, whereas ΔpilA[1-6] strains expressing PilA3His or PilA4His adhere even more strongly than the parental strain. Most surprisingly, expressing either PilA5His or PilA6His in the ΔpilA[1-6] strain results in microcolony formation. A hybrid protein in which the conserved N terminus of the mature PilA1His is replaced with the corresponding N domain of FlgA1 is processed by the prepilin peptidase, but it does not assemble functional pili, leading us to conclude that Duf1628 can be annotated as the N terminus of archaeal PilA adhesion pilins. Finally, the pilin prediction program, FlaFind, which was trained primarily on archaeal flagellin sequences, was successfully refined to more accurately predict pilins based on the in vivo verification of PilA[1-6].