Unsupervised Online Paired Associates Learning Task from the Cambridge Neuropsychological Test Automated Battery (CANTAB®) in the Brain Health Registry.

BACKGROUND: Unsupervised online cognitive assessments have demonstrated promise as an efficient and scalable approach for evaluating cognition in aging, and Alzheimer's disease and related dementias. OBJECTIVES: The aim of this study was to evaluate the feasibility, usability, and construct validity of the Paired Associates Learning task from the Cambridge Neuropsychological Test Automated Battery® in adults enrolled in the Brain Health Registry. DESIGN, SETTING, PARTICIPANTS, MEASUREMENTS: The Paired Associates Learning task was administered to Brain Health Registry participants in a remote, unsupervised, online setting. In this cross-sectional analysis, we 1) evaluated construct validity by analyzing associations between Paired Associates Learning performance and additional participant registry data, including demographics, self- and study partner-reported subjective cognitive change (Everyday Cognition scale), self-reported memory concern, and depressive symptom severity (Patient Health Questionnaire-9) using multivariable linear regression models; 2) determined the predictive value of Paired Associates Learning and other registry variables for identifying participants who self-report Mild Cognitive Impairment by employing multivariable binomial logistic regressions and calculating the area under the receiver operator curve; 3) investigated feasibility by looking at task completion rates and statistically comparing characteristics of task completers and non-completers; and 4) evaluated usability in terms of participant requests for support from BHR related to the assessment. RESULTS: In terms of construct validity, in participants who took the Paired Associates Learning for the first time (N=14,528), worse performance was associated with being older, being male, lower educational attainment, higher levels of self- and study partner-reported decline, more self-reported memory concerns, greater depressive symptom severity, and self-report of Mild Cognitive Impairment. Paired Associates Learning performance and Brain Health Registry variables together identified those with self-reported Mild Cognitive Impairment with moderate accuracy (areas under the curve: 0.66-0.68). In terms of feasibility, in a sub-sample of 29,176 participants who had the opportunity to complete Paired Associates Learning for the first time in the registry, 14,417 started the task. 11,647 (80.9% of those who started) completed the task. Compared to those who did not complete the task at their first opportunity, those who completed were older, had more years of education, more likely to self-identify as White, less likely to self-identify as Latino, less likely to have a subjective memory concern, and more likely to report a family history of Alzheimer's disease. In terms of usability, out of 8,395 received requests for support from BHR staff via email, 4.4% (n=374) were related to PAL. Of those, 82% were related to technical difficulties. CONCLUSIONS: Our findings support moderate feasibility, good usability, and construct validity of cross-sectional Paired Associates Learning in an unsupervised online registry, but also highlight the need to make the assessment more inclusive and accessible to individuals from ethnoculturally and socioeconomically diverse communities. A future, improved version could be a scalable, efficient method to assess cognition in many different settings, including clinical trials, observational studies, healthcare, and public health.

Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding.

Like other enveloped viruses, HIV-1 uses cellular machinery to bud from infected cells. We now show that Tsg101 protein, which functions in vacuolar protein sorting (Vps), is required for HIV-1 budding. The UEV domain of Tsg101 binds to an essential tetrapeptide (PTAP) motif within the p6 domain of the structural Gag protein and also to ubiquitin. Depletion of cellular Tsg101 by small interfering RNA arrests HIV-1 budding at a late stage, and budding is rescued by reintroduction of Tsg101. Dominant negative mutant Vps4 proteins that inhibit vacuolar protein sorting also arrest HIV-1 and MLV budding. These observations suggest that retroviruses bud by appropriating cellular machinery normally used in the Vps pathway to form multivesicular bodies.

dnaC mutations suppress defects in DNA replication- and recombination-associated functions in priB and priC double mutants in Escherichia coli K-12.

PriA, PriB and PriC were originally discovered as proteins essential for the PhiX174 in vitro DNA replication system. Recent studies have shown that PriA mutants are poorly viable, have high basal levels of SOS expression (SOSH), are recombination deficient (Rec-), sensitive to UV irradiation (UVS) and sensitive to rich media. These data suggest that priA's role may be more complex than previously thought and may involve both DNA replication and homologous recombination. Based on the PhiX174 system, mutations in priB and priC should cause phenotypes like those seen in priA2:kan mutants. To test this, mutations in priB and priC were constructed. We found that, contrary to the PhiX174 model, del(priB)302 and priC303:kan mutants have almost wild-type phenotypes. Most unexpectedly, we then found that the priBC double mutant had very poor viability and/or a slow growth rate (even less than a priA2:kan mutant). This suggests that priB and priC have a redundant and important role in Escherichia coli. The priA2:kan suppressor, dnaC809, partially suppressed the poor viability/slow growth phenotype of the priBC double mutant. The resulting triple mutant (priBC dnaC809 ) had small colony size, recombination deficiency and levels of SOS expression similar to a priA2:kan mutant. The priBC dnaC809 mutant, however, was moderately UVR and had good viability, unlike a priA2:kan mutant. Additional mutations in the triple mutant were selected to suppress the slow growth phenotype. One suppressor restored all phenotypes tested to nearly wild-type levels. This mutation was identified as dnaC820 (K178N) [mapping just downstream of dnaC809 (E176G)]. Experiments suggest that dnaC820 makes dnaC809 suppression of priA and or priBC mutants priB and or priC independent. A model is proposed for the roles of these proteins in terms of restarting collapsed replication forks from recombinational intermediates.

ebi regulates epidermal growth factor receptor signaling pathways in Drosophila.

ebi regulates the epidermal growth factor receptor (EGFR) signaling pathway at multiple steps in Drosophila development. Mutations in ebi and Egfr lead to similar phenotypes and show genetic interactions. However, ebi does not show genetic interactions with other RTKs (e.g., torso) or with components of the canonical Ras/MAP kinase pathway. ebi encodes an evolutionarily conserved protein with a unique amino terminus, distantly related to F-box sequences, and six tandemly arranged carboxy-terminal WD40 repeats. The existence of closely related proteins in yeast, plants, and humans suggests that ebi functions in a highly conserved biochemical pathway. Proteins with related structures regulate protein degradation. Similarly, in the developing eye, ebi promotes EGFR-dependent down-regulation of Tramtrack88, an antagonist of neuronal development.

roughex down-regulates G2 cyclins in G1.

Cell cycle arrest in G1 at the onset of patterning in the Drosophila eye is mediated by roughex. In roughex mutants, cells accumulate Cyclin A protein in early G1 and progress into S phase precociously. When Roughex is overexpressed in S/G2 cells, Cyclin A is mislocalized to the nucleus and degraded, preventing mitosis. Whereas Roughex inhibits Cyclin A accumulation, Cyclin E down-regulates Roughex protein in vivo. Roughex binds to Cyclin E and is a substrate for a Cyclin E-Cdk complex in vitro. These data argue that Roughex inhibits Cyclin A accumulation in early G1 by targeting Cyclin A for destruction. In late G1, Roughex is destabilized in a Cyclin E-dependent process, releasing Cyclin A for its role in S/G2.

Control of G1 in the developing Drosophila eye: rca1 regulates Cyclin A.

In the developing eye of Drosophila melanogaster, cells become synchronized in the G1 phase of the cell cycle just prior to the onset of cellular differentiation and morphogenesis. In roughex (rux) mutants, cells enter S phase precociously because of ectopic activation of a Cyclin A/Cdk complex in early G1. This leads to defects in cell fate and pattern formation, and results in abnormalities in the morphology of the adult eye. A screen for dominant suppressors of the rux eye phenotype led to the identification of mutations in cyclin A, string (cdc25), and new cell cycle genes. One of these genes, regulator of cyclin A (rca1), encodes a novel protein required for both mitotic and meiotic cell cycle progression. rca1 mutants arrest in G2 of embryonic cell cycle 16 with a phenotype very similar to cyclin A loss of function mutants. Expression of rca1 transgenes in G1 or in postmitotic neurons promotes Cyclin A protein accumulation and drives cells into S phase in a Cyclin A-dependent fashion.

The eye-specification proteins So and Eya form a complex and regulate multiple steps in Drosophila eye development.

Sine oculis (so) and eyes absent (eya) are required for Drosophila eye development and are founding members of the mammalian Six and Eya gene families. These genes have been proposed to act with eyeless (Pax6) to regulate eye development in vertebrates and invertebrates. so encodes a highly diverged homeobox transcription factor and eya encodes a novel nuclear protein. We demonstrate that So and Eya (1) regulate common steps in eye development including cell proliferation, patterning, and neuronal development; (2) synergize in inducing ectopic eyes; and (3) interact in yeast and in vitro through evolutionarily conserved domains. We propose that an So/Eya complex regulates multiple steps in eye development and functions within the context of a network of genes to specify eye tissue identity.

Controlling cell proliferation in differentiating tissues: genetic analysis of negative regulators of G1-->S-phase progression.

Withdrawal from the cell cycle as cells begin to differentiate is accomplished by the downregulation of cyclin-dependent kinase activities in G1 phase. Recent analysis of loss-of-function mutations in flies, worms, and mice has provided insight into the roles of various negative regulators of G1 phase in developing organisms.

The Drosophila rolled locus encodes a MAP kinase required in the sevenless signal transduction pathway.

Mitogen-activated protein (MAP) kinases have been proposed to play a critical role in receptor tyrosine kinase (RTK)-mediated signal transduction pathways. Although genetic and biochemical studies of RTK pathways in Caenorhabditis elegans, Drosophila melanogaster and mammals have revealed remarkable similarities, a genetic requirement for MAP kinases in RTK signaling has not been established. During retinal development in Drosophila, the sevenless (Sev) RTK is required for development of the R7 photoreceptor cell. Components of the signal transduction pathway activated by Sev in the R7 precursor include proteins encoded by the gap1, drk, Sos, ras1 and raf loci. In this report we present evidence that a Drosophila MAP kinase, ERK-A, is encoded by the rolled locus and is required downstream of raf in the Sev signal transduction pathway.

Helicase-deficient cysteine to glycine substitution mutants of Escherichia coli replication protein PriA retain single-stranded DNA-dependent ATPase activity. Zn2+ stimulation of mutant PriA helicase and primosome assembly activities.

The PriA replication protein of Escherichia coli guides the ordered assembly of the primosome, the mobile, multiprotein, bidirectional, DNA replication priming/helicase complex of which it is an integral part. Although the PriA protein is not essential for viability, primosome assembly via a PriA-dependent pathway is required for normal cellular replication and growth. The PriA protein itself is multifunctional. In addition to its role in directing primosome assembly, PriA is a site-specific, single-stranded DNA-dependent ATPase (dATPase) and a 3'-->5' DNA translocase and helicase. In an attempt to assess how each individual PriA activity is related to the others (i.e. can one activity function independently of the others or are they intrinsically coupled?), a series of site-directed mutations within priA have been created. priA encodes a cysteine-rich motif, the sequence of which suggests that this region of the protein may be involved in metal binding. Biochemical characterization of three purified cysteine to glycine substitution mutant PriA proteins revealed that these mutant proteins retained their site-specific single-stranded DNA-dependent ATPase activity. However, two of the three mutant proteins were completely incapable of any helicase activity. Residual helicase activity of the third mutant PriA protein could be stimulated 3-fold by the presence of low concentrations of Zn2+ ions. Primosomes assembled with the mutant PriA proteins were also defective in both their ability to act as bidirectional helicase complexes, as well as their ability to synthesize primers for extension by the DNA polymerase III holoenzyme. The results presented here suggest that the cysteine-rich region of PriA is indeed involved in metal binding and that single cysteine to glycine substitutions within this region result in the uncoupling of the ATPase activity of PriA from both its helicase activity and its ability to interact correctly with the DNA template and the six other primosomal proteins.

ATPase-deficient mutants of the Escherichia coli DNA replication protein PriA are capable of catalyzing the assembly of active primosomes.

The PriA replication protein of Escherichia coli (formerly replication factor Y or protein n') is multifunctional. It is a site-specific, single-stranded DNA-dependent ATPase (dATPase), a 3'----5' DNA helicase, and guides the ordered assembly of the primosome, a mobile, multiprotein DNA replication priming/helicase complex. Although PriA is not absolutely required for viability, priA null mutant cells grow very slowly, have poor viability, and form extensive filaments. In order to assess which of the multiple activities of PriA are required for normal replication and growth, site-directed mutagenesis was employed to introduce single amino acid substitutions for the invariant lysine within the consensus nucleotide-binding motif found in PriA. Biochemical characterization of the representative purified mutant PriA proteins revealed them to be completely deficient in nucleotide hydrolysis, incapable of translocation along a single-stranded DNA binding protein-coated single-stranded DNA template, and unable to manifest the 3'----5' DNA helicase activity of wild-type PriA. These mutant proteins were, however, capable of catalyzing the assembly of active primosomes in vitro. Furthermore, when supplied in trans to insertionally inactivated priA cells, plasmids containing a copy of these mutant priA genes restored the wild-type growth rate, viability, and cell morphology. Based on these results, a model for PriA function in vivo is discussed.

Dissecting the functional role of PriA protein-catalysed primosome assembly in Escherichia coli DNA replication.

The multi-functional PriA protein of Escherichia coli (formerly replication factor Y or protein n') serves to guide the ordered assembly of the primosome, a mobile multiprotein replication priming/helicase complex. Primosome assembly is essential for bacteriophage OX174 complementary DNA strand synthesis and ColE1-type plasmid replication reconstituted in vitro with purified proteins. The biochemical activities of the primosome suggest that it can fulfill the primase/helicase requirement on the lagging-strand DNA template during cellular DNA replication. However, reconstruction in vitro of DNA replication of small plasmids containing the E. coli origin of DNA replication (oriC) does not require the complete complement of primosomal proteins. Thus, the extent to which PriA-catalysed primosome assembly participates in chromosomal replication has remained unclear. The recent isolation of the genes encoding PriA, PriB (protein n), PriC (protein n"), and DnaT (protein i) has provided the necessary tools for addressing this issue. The phenotype of mutations in these genes, and other results described in this review, suggest that assembly of the primosome catalysed by PriA does in fact contribute at some stage to normal cellular DNA replication. A model for primososme-catalysed reactivation of a dysfunctional replication fork is discussed.

Inactivation of the Escherichia coli priA DNA replication protein induces the SOS response.

Many of the proteins that operate at the replication fork in Escherichia coli have been defined genetically. These include some of the subunits of the DNA polymerase III holoenzyme, the DnaB replication fork helicase, and the DnaG primase. The multiprotein primosome (which includes the DnaB and DnaG proteins), defined biochemically on the basis of its requirement during bacteriophage phi X174 complementary-strand synthesis, could serve as the helicase-primase replication machine on the lagging-strand template. In order to determine if this is the case, we have begun an investigation of the phenotypes of mutants with mutations priA, priB, and priC, which encode the primosomal proteins factor Y (protein n'), n, and n", respectively. Inactivation of priA by insertional mutagenesis resulted in the induction of the SOS response, as evinced by induction of a resident lambda prophage, extreme filamentation, and derepression of an indicator operon in which beta-galactosidase production was controlled by the dinD1 promoter. In addition, the copy numbers of resident pBR322 plasmids were reduced four- to fivefold in these strains, and production of phi X174 phage was delayed considerably. These results are discussed in the context of existing models for SOS induction and possible roles for the PriA protein at the replication fork in vivo.

The priB and priC replication proteins of Escherichia coli. Genes, DNA sequence, overexpression, and purification.

The Escherichia coli DNA replication proteins n and n" function in vitro in the assembly of the primosome, a mobile multiprotein replication priming complex thought to operate on the lagging-strand template at the E. coli DNA replication fork. Both proteins have been purified from E. coli HMS83 cells based on their requirement for the reconstitution of bacteriophage phi X174 complementary strand DNA synthesis in vitro with purified proteins. As a step toward understanding the role of these proteins in vivo, the genes for primosomal proteins n and n", designated priB and priC, respectively, have been cloned molecularly. priB encodes a 104-amino acid 11.4-kDa polypeptide and corresponds to an previously identified open reading frame between rpsF and rps R within a ribosomal protein operon at 95.5 min on the E. coli chromosome. priC encodes a 175-amino acid 20.3-kDa polypeptide. These two gene products were overexpressed at least 1000-fold in E. coli using a bacteriophage T7 transient expression system. Both proteins have been purified to apparent homogeneity from extracts prepared from these overproducing strains.

Molecular cloning and DNA sequence analysis of Escherichia coli priA, the gene encoding the primosomal protein replication factor Y.

Escherichia coli replication factor Y (protein n') functions in the assembly of a mobile multiprotein replication-priming complex called the primosome. Although the role of factor Y in primosome assembly during replication in vitro of bacteriophage phi X174 and plasmid pBR322 DNA is clear, its role in E. coli chromosomal replication is not. To address this issue, the gene for factor Y has been cloned molecularly and its DNA sequence has been determined. The cloned fragment of DNA contained an open reading frame capable of encoding a polypeptide of 81.7 kDa. This open reading frame contains amino acid sequences identical to 13 N-terminal amino acids of purified factor Y, as well as to a 10-amino acid internal sequence (from a cyanogen bromide fragment) as determined by gas-phase microsequencing. Expression of the polypeptide encoded by this open reading frame using a bacteriophage T7 transient expression system resulted in the accumulation of a polypeptide with an apparent molecular mass of 78 kDa that comigrated with bona fide factor Y during SDS/polyacrylamide gel electrophoresis. Soluble extracts made from cells overexpressing the product of the putative factor Y open reading frame showed a 2000-fold increase in factor Y activity during bacteriophage phi X174 complementary-strand DNA synthesis in vitro when compared to control extracts. The gene encoding factor Y, which maps to 88.5 min on the E. coli chromosome, has been designated primosome A (priA).