Abstract. ot-Lytic protease is a bacterial serine protease of the trypsin family that is synthesized as a 39-kD preproenzyme (Silen, J. L., C. N. McGrath, K. R. Smith, and D. A. Agard. 1988. Gene (Amst.). 69: 23%244). The 198-amino acid mature protease is secreted into the culture medium by the native host, Lysobacter enzymogenes (Whitaker, D. R. 1970. Methods Enzymol. 19:599-613). Expression experiments in Escherichia coli revealed that the 166-amino acid pro region is transiently required either in cis ( Here we show that full-length precursor produced at nonpermissive temperatures is tightly associated with the E. coli outer membrane. The active site mutant Ser 195--'Ala (SA195), which is incapable of self-processing, also accumulates as a precursor in the outer membrane, even when expressed at permissive temperatures. When the protease domain is expressed in the absence of the pro region, the misfolded, inactive protease also cofractionates with the outer membrane. However, when the folding requirement for either wild-type or mutant protease domains is provided by expressing the pro region in trans, both are efficiently secreted into the extracellular medium. Attempts to separate folding and secretion functions by extensive deletion mutagenesis within the pro region were unsuccessful. Taken together, these results suggest that only properly folded and processed forms of ot-lytic protease are efficiently transported to the medium.

Rep protein and helicase IV, two DNA-dependent adenosine 5'-triphosphatases with helicase activity, have been purified from Escherichza coli and characterized. Both enzymes exhibit a distributive interaction with single-stranded DNA as DNA-dependent ATPases in a reaction that is relatively resistant to increasing NaCl concentration and sensitive to the addition of E. coli single-stranded DNA binding protein (SSB). The helicase reaction catalyzed by each protein has been characterized using a direct unwinding assay and partial duplex DNA substrates. Both Rep protein and helicase IV catalyzed the unwinding of a duplex region 71 bp in length. However, unwinding of a 119-bp or 343-bp duplex region was substantially reduced compared to unwinding of the 71-bp substrate. At each concentration of protein examined, the number of base pairs unwound was greatest using the 71-bp substrate, intermediate with the 119-bp substrate and lowest using the 343-bp substrate. The addition of E. coli SSB did not increase the fraction of the 343-nucleotide fragment unwound by Rep protein. However, the addition of SSB did stimulate the unwinding reaction catalyzed by helicase IV approximately twofold. In addition, ionic strength conditions which stabilize duplex DNA (i.e. addition of MgC12 or NaCl), markedly inhibited the helicase reaction catalyzed by either Rep protein or helicase IV while having little effect on the ATPase reaction. Thus, these two enzymes appear to share a common biochemical mechanism for unwinding duplex DNA which can be described as limited unwinding of duplex DNA. Taken together these data suggest that, in vitro, and in the absence of additional proteins, neither Rep protein nor helicase IV catalyzes a processive unwinding reaction.

BackgroundAmyloid‐PET had been widely used and validated in research settings, using highly selected samples, harmonized acquisition protocols, co‐registration with MRI, and central interpretation by highly experienced experts. In contrast, in clinical settings, more heterogeneous acquisition, reconstruction, and interpretation may compromise the accuracy of the imaging. We quantitatively analyzed real‐world amyloid‐PET scans to assess their validity.MethodIDEAS acquired 18,295 amyloid PET scans at 343 PET facilities in patients with MCI or dementia using 18F‐florbetapir, 18F‐florbetaben, or 18F‐flutemetamol. Scans were visually interpreted at each site as either negative or positive for cortical tracer retention. Scans from consenting patients were archived. As of December 1, 2021, amyloid‐PET scans from 6,263 unique participants were available for analysis. Exclusion for lack of valid images or clinical data and failure of quality checks resulted in 6,150 (98.2%) valid scans. We analyzed the scans using a recently validated PET‐only processing pipeline designed to process heterogeneous amyloid‐PET scans (Iaccarino et al, 2022) and quantified cortical uptake in Centiloid (CL) units. A previously established neuropathology‐based threshold of 24.4 CL was used to define amyloid‐PET positivity independent of visual reads.ResultMean CL was higher in dementia (mean±SD = 53±51) than in MCI (40±48) (mean difference: 13; 95%CI: 10‐15). Mean CL of visually negative scans (3±27) was very close to 0, as expected for patients without amyloid accumulation, and significantly lower than visually positive scans (72±41) (mean difference: 69; 95%CI: 67‐70) (table 1). High concordance was found between local visual reads and CL‐based positivity (86.5%, Cohen’s κ=0.72, figure 1). CL exhibited a bimodal distribution, with most scans clearly positive or negative, and a minority of visual‐quantitative discordant scans surrounding the positivity threshold (figure 2). CL negatively correlated with MMSE (r=‐0.19, p<.001, figure 3). CL further correlated with the level of confidence in the diagnosis of Alzheimer’s Disease (AD), as was indicated by clinicians before the performance of PET (r=0.13, p<.001, figure 4).ConclusionA large heterogeneous dataset of real‐world amyloid‐PET scans analyzed quantitatively, shows high concordance with visual reads, and expected relationships with clinical and neuropsychological measures of AD.

Background: Imaging biomarkers for neurodegenerative disease are understudied in diverse populations. The completed Imaging Dementia-Evidence for Amyloid Scanning (IDEAS) and recently launched New IDEAS studies evaluate the clinical impact of amyloid PET in large samples of Medicare beneficiaries across the U.S. with MCI or dementia, enabling comparisons between patients from different racial and ethnic backgrounds. Method: Amyloid PET was performed in 18,295 IDEAS Study participants recruited from 590 clinical sites between 2016-2018. Dementia specialists completed pre-and post-PET case report forms. Medicare claims were followed for 12 months. New IDEAS will enroll 7000 Medicare beneficiaries (minimum 2000 African Americans and 2000 Latinos) with MCI or dementia at ∼350 memory clinics across the US between 2020-2023, following analogous procedures. Quantified social determinants of health (quality of education, midlife/current income, marital status and living arrangement) will be measured. A team involving Vanderbilt University, University of North Carolina and the Alzheimer's Association will use community engaged research principles (community stakeholders as study champions, tailored recruitment strategies) to ensure cohort diversity.Result: Recruitment in IDEAS included a low proportion of minority participants (83.9% White (N=15,568), 3.4% African American (N=639), 4.6% Hispanic (N=848), 1.8% Asian (N=328)). The proportion of positive amyloid scans was lower for African Americans (53.8%), Hispanics (54%) and Asians (45.1%) compared to Whites (62.7%).African Americans (52%), Hispanics (56%) and Asians (47%) presented more frequently with dementia (as opposed to MCI) than Whites (37%). There were disparities in vascular risk factors (hypertension/diabetes: 68%/28% African Americans, 55%/26% Hispanics, 48%/25% Asians, 50%/15% Whites) and lower educational attainment for African Americans and Hispanics. New IDEAS will examine the impact of amyloid PET on health outcomes and patient management in the total cohort and

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