Cell-free DNA-based prenatal screening via rolling circle amplification: Identifying and resolving analytic issues.

OBJECTIVE: A rolling circle amplification (RCA) based commercial methodology using cell-free (cf)DNA to screen for common trisomies became available in 2018. Relevant publications documented high detection but with a higher than expected 1% false positive rate. Preliminary evidence suggested assay variability was an issue. A multi-center collaboration was created to explore this further and examine whether subsequent manufacturer changes were effective. METHODS: Three academic (four devices) and two commercial (two devices) laboratories provided run date, chromosome 21, 18, and 13 run-specific standard deviations, number of samples run, and reagent lot identifications. Temporal trends and between-site/device consistency were explored. Proportions of run standard deviations exceeding pre-specified caps of 0.4%, 0.4% and 0.6% were computed. RESULTS: Overall, 661 RCA runs between April 2019 and July 30, 2022 tested 39,756 samples. In the first 24, subsequent 9, and final 7 months, proportions of capped chromosome 21 runs dropped from 39% to 22% to 6.0%; for chromosome 18, rates were 76%, 36%, and 4.0%. Few chromosome 13 runs were capped using the original 0.60%, but capping at 0.50%, rates were 28%, 16%, and 7.6%. Final rates occurred after reformulated reagents and imaging software modifications were fully implemented across all devices. Revised detection and false positive rates are estimated at 98.4% and 0.3%, respectively. After repeat testing, failure rates may be as low as 0.3%. CONCLUSION: Current RCA-based screening performance estimates are equivalent to those reported for other methods, but with a lower test failure rate after repeat testing.

Kinesin-1 tail autoregulation and microtubule-binding regions function in saltatory transport but not ooplasmic streaming.

The N-terminal head domain of kinesin heavy chain (Khc) is well known for generating force for transport along microtubules in cytoplasmic organization processes during metazoan development, but the functions of the C-terminal tail are not clear. To address this, we studied the effects of tail mutations on mitochondria transport, determinant mRNA localization and cytoplasmic streaming in Drosophila. Our results show that two biochemically defined elements of the tail - the ATP-independent microtubule-binding sequence and the IAK autoinhibitory motif - are essential for development and viability. Both elements have positive functions in the axonal transport of mitochondria and determinant mRNA localization in oocytes, processes that are accomplished by biased saltatory movement of individual cargoes. Surprisingly, there were no indications that the IAK autoinhibitory motif acts as a general downregulator of Kinesin-1 in those processes. Time-lapse imaging indicated that neither tail region is needed for fast cytoplasmic streaming in oocytes, which is a non-saltatory bulk transport process driven solely by Kinesin-1. Thus, the Khc tail is not constitutively required for Kinesin-1 activation, force transduction or linkage to cargo. It might instead be crucial for more subtle elements of motor control and coordination in the stop-and-go movements of biased saltatory transport.

Non-specific interference in the measurement of plasma ammonia: importance of using a sample blank.

BACKGROUND: Enzymatic assays using glutamate dehydrogenase (GLDH) to monitor the transformation of NAD(P)H to NAD(P)(+) by a spectrophotometric technique are the most common methods to measure plasma ammonia (PA) in routine laboratories worldwide. However, these assays can potentially be subject to interference by substances in plasma able to oxidize NAD(P)H at a substantial rate, thereby providing falsely high results. METHODS: To study this potential interference, we spiked a plasma pool with a liver homogenate and measured the ammonia concentration using a dry chemistry system (Vitros 250, Ortho Clinical Diagnostic, Raritan, NJ, USA), an enzymatic assay without a sample blanking step (Infinity Ammonia Liquid Stable Reagent, Thermo Fisher Scientific, Waltham, USA) and an enzymatic assay that corrects for the non-specific oxidation of NADPH (Ammonia kit, RANDOX Laboratories Ltd, Crumlin, UK). RESULTS: This experiment shows that the Infinity ammonia reagent kit is subject to a clinically significant interference and explains the discrepancies previously reported between these methods in patients with acute liver failure (ALF). CONCLUSION: When using enzymatic methods for the assessment of PA, we recommend including a sample blanking correction and this should be mandatory when monitoring patients with ALF.

A population-based epidemiologic study of Helicobacter pylori infection and its association with systemic inflammation.

BACKGROUND: Infection with Helicobacter pylori is associated with a variety of non-gastrointestinal sequelae. These may be mediated by an increase in systemic inflammation. We assessed if serologic evidence of infection with H. pylori is associated with increased serum C-reactive protein (CRP) levels. METHODS: The study design consisted of a randomly selected, cross-sectional population-based study of 2633 individuals phenotyped in 1991, of whom 2361 participants provided serum samples to permit measurement of H. pylori's serologic status and CRP levels. RESULTS: Male gender (odds ratio (OR): 1.65; 95% confidence interval (CI): 1.23-2.21), age (OR per year: 1.05; 95% CI: 1.04-1.06), height (OR per meter: 0.05; 95% CI: 0.01-0.24), current smoking habit (compared with never smokers, OR: 1.46; 95% CI: 1.13-1.88), and less affluent socioeconomic status were associated with increased odds of being seropositive for H. pylori. Helicobacter pylori infection was associated with increased risk of having an elevated serum CRP (above 3 mg/L) after adjustment for gender, age, height, smoking status, and socioeconomic status (OR: 1.32; 95% CI: 1.05-1.67). Similar associations were seen using a threshold for elevated serum CRP of greater than 1 mg/L. CONCLUSIONS: Our data suggest that infection with H. pylori is associated with increased systemic inflammation. This suggests one potential mechanism to explain the extra-gastrointestinal conditions associated with H. pylori infection.

Helicobacter pylori and lung function, asthma, atopy and allergic disease--a population-based cross-sectional study in adults.

BACKGROUND: Exposure to microbes may result in the polarization of the immune system and a decrease in the risk of asthma and associated allergic disease, whilst exposure to Helicobacter pylori has been hypothesized to increase the risk of obstructive airways disease. We tested the hypotheses that exposure to H. pylori reduces the risk of asthma and allergic disease and is associated with a decrease in lung function. METHODS: Data were collected on allergic disease symptoms, forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC), bronchial reactivity, allergen skin sensitization, serum IgE and H. pylori serological status in 2437 randomly selected adults. RESULTS: Individuals with serological evidence of exposure to H. pylori had lower lung function, FEV1 being lower by 53 ml (95% CI 1-106) and FVC 83 ml (95% CI 20-145) lower in the cross-sectional analysis. These differences ceased to be statistically significant after adjustment for height or socio-economic status. There was no association between H. pylori serological status and measures of asthma or atopy in the cross-sectional analysis, and there was no significant association between H. pylori serological status and decline in FEV1 and FVC over 9 years. CONCLUSION: Although H. pylori exposure may be associated with lower cross-sectional FEV1 and FVC, this association was not independent of height or socio-economic status. There was no association between H. pylori exposure and either chronic obstructive pulmonary disease (COPD), measures of allergic disease or decline in lung function.

Coordinated activation of Hsp70 chaperones.

Hsp70s are a ubiquitous family of molecular chaperones involved in many cellular processes. Two Hsp70s, Lhs1p and Kar2p, are required for protein biogenesis in the yeast endoplasmic reticulum. Here, we found that Lhs1p and Kar2p specifically interacted to couple, and coordinately regulate, their respective activities. Lhs1p stimulated Kar2p by providing a specific nucleotide exchange activity, whereas Kar2p reciprocally activated the Lhs1p adenosine triphosphatase (ATPase). The two ATPase activities are coupled, and their coordinated regulation is essential for normal function in vivo.