An Approach for Evaluating Potential Screening Thresholds Using Biomarker Population Distribution and Analytical Imprecision.

BACKGROUND: A common approach in laboratory medicine is to use a simple but sensitive test to screen samples to identify those that require additional investigation with a more complex and informative method. Selection of screening thresholds can be guided by biomarker distribution in the tested population and the analytical imprecision of the method. METHODS: A simulation using joint probabilities derived from the population distribution for galactose-1-phosphate uridylyltransferase (GALT) activity and the analytical imprecision for the GALT assay was used to estimate the number of samples that would require repeat analysis and the number of samples with possibly false-negative screening determinations due to analytical imprecision. RESULTS: In the case of GALT activity, screening a conservative initial threshold 6 standard deviations from the confirmation threshold can essentially eliminate the chance of a false-negative screening determination due to analytical imprecision. The trade-off is a greater number of samples requiring follow-up testing (n = 222, equivalent to 0.15% of samples annually). CONCLUSIONS: Selection of thresholds in a screening algorithm is informed by estimates of the number of samples that would require repeat testing and the number that could be false negative due to analytical imprecision.

Biotinidase activity is affected by both seasonal temperature and filter collection cards.

This study set out to examine pre-analytical factors affecting the frequency of positive results in newborn screening for biotinidase deficiency. This investigation was prompted by an increase in the annual screen positive rate for biotinidase deficiency in Ontario from 2.65x10-4 in 2016 to 6.57x10-4 in 2017. Season and trend decomposition was used to separate seasonality from an underlying trend in the time series of biotindase activity measurements for the period 2014-01-12 to 2019-07-27 (n = 798,770). This analysis revealed a marked seasonal effect (winter = median + ⩽ 17 MRU, summer = mean - ⩽20 MRU) and a non-linear negative trend. Seasonal temperature was correlated with biotinidase results (Pearson's r = 0.79) but not with the observed negative trend (Pearson's r = 0.0025). Time series analysis of biotinidase results grouped by print lot of filter paper revealed that recently printed filter paper cards inhibit biotinidase and that this inhibition resolved over time. This study demonstrates that biotindase activity is inhibited by both increased seasonal temperature and collection on newly printed filter cards.

Stability and function of the Sec61 translocation complex depends on the Sss1p tail-anchor sequence.

Sss1p, an essential component of the heterotrimeric Sec61 complex in the ER (endoplasmic reticulum), is a tail-anchored protein whose precise mechanism of action is largely unknown. Tail-anchored proteins are involved in many cellular processes and are characterized by a single transmembrane sequence at or near the C-terminus. The Sec61 complex is the molecular machine through which secretory and membrane proteins translocate into and across the ER membrane. To understand the function of the tail anchor of Sss1p, we introduced mutations into the tail-anchor sequence and analysed the resulting yeast phenotypes. Point mutations in the C-terminal hydrophobic core of the tail anchor of Sss1p were identified that allowed Sss1p assembly into Sec61 complexes, but resulted in diminished growth, defects in co- and post-translational translocation, inefficient ribosome binding to Sec61 complexes, reduction in the stability of both heterotrimeric Sec61 and heptameric Sec complexes and a complete breakdown of ER structure. The underlying defect caused by the mutations involves loss of a stabilizing function of the Sss1p tail-anchor sequence for both the heterotrimeric Sec61 and the heptameric Sec complexes. These results indicate that by stabilizing multiprotein membrane complexes, the hydrophobic core of a tail-anchor sequence can be more than a simple membrane anchor.

Unexpectedly low pulse oximetry measurements associated with variant hemoglobins: a systematic review.

Pulse oximetry estimates arterial blood oxygen saturation based on light absorbance of oxy- and deoxy-hemoglobin at 660 and 940 nm wavelengths. Patients with unexpectedly low SpO2 often undergo cardio-pulmonary testing to ascertain the cause of their hypoxemia. However, in a subset of patients, a variant hemoglobin is responsible for low SpO2 measurements. The extent of this problem is unclear. We performed a systematic literature review for reports of low SpO2 associated with variant hemoglobins. We also reviewed unpublished cases from an academic hemoglobin diagnostic reference laboratory. Twenty-five publications and four unpublished cases were identified, representing 45 patients with low SpO2 and confirmed variant hemoglobin. Fifty-seven family members of patients had confirmed or suspected variant hemoglobin. Three low oxygen affinity variant hemoglobins had concordantly low SpO2 and SaO2. Eleven variant hemoglobins were associated with unexpectedly low SpO2 measurements but normal SaO2. Hemoglobin light absorbance testing was reported in three cases, all of which showed abnormal absorption spectra between 600 and 900 nm. Seven other variant hemoglobins had decreased SpO2, with unreported or uncertain SaO2. Twenty-one variant hemoglobins were found to be associated with low SpO2. Most variant hemoglobins were associated with spuriously low SpO2. Abnormal absorption spectra explain the discrepancy between SpO2 and SaO(2) for some variants. The differential diagnosis of possible variant hemoglobin ought to be considered in asymptomatic patients found to have unexpectedly low SpO2. The correct diagnosis will help to spare patients from unnecessary investigations and anxiety.

The C-terminus of cytochrome b5 confers endoplasmic reticulum specificity by preventing spontaneous insertion into membranes.

The molecular mechanisms that determine the correct subcellular localization of proteins targeted to membranes by tail-anchor sequences are poorly defined. Previously, we showed that two isoforms of the tung oil tree [Vernicia (Aleurites) fordii] tail-anchored Cb5 (cytochrome b5) target specifically to ER (endoplasmic reticulum) membranes both in vivo and in vitro [Hwang, Pelitire, Henderson, Andrews, Dyer and Mullen (2004) Plant Cell 16, 3002-3019]. In the present study, we examine the targeting of various tung Cb5 fusion proteins and truncation mutants to purified intracellular membranes in vitro in order to assess the importance of the charged CTS (C-terminal sequence) in targeting to specific membranes. Removal of the CTS from tung Cb5 proteins resulted in efficient binding to both ER and mitochondria. Results from organelle competition, liposome-binding and membrane proteolysis experiments demonstrated that removal of the CTS results in spontaneous insertion of tung Cb5 proteins into lipid bilayers. Our results indicate that the CTSs from plant Cb5 proteins provide ER specificity by preventing spontaneous insertion into incorrect subcellular membranes.

Cytokines and cell adhesion molecules exhibit distinct profiles in health, ovarian cancer, and breast cancer.

OBJECTIVE: We examined a panel of cytokines and cell adhesion molecules in an attempt to identify cancer specific profiles. DESIGN AND METHODS: Cytokines and cell adhesion arrays (Randox Ltd.) were measured in samples from women with a histological diagnosis of ovarian cancer ([Formula: see text]) or breast cancer ([Formula: see text]) or cancer free ([Formula: see text]). Random forest analysis was used for classification. RESULTS: Ovarian cancer subjects were classified with a sensitivity of 85.7% (95% CI 50-100) and a specificity of 84.2% (95% CI 69.4-93.4). Breast cancer subjects were classified with a sensitivity of 70.8% (95% CI 47.1-86.4) and a specificity of 96.4% (95% CI 82.1-100). DISCUSSION: Cytokine and cell adhesion molecule profiles provide additional information that may be useful for cancer characterization of female cancers.

Astrocytes Surviving Severe Stress Can Still Protect Neighboring Neurons from Proteotoxic Injury.

Astrocytes are one of the major cell types to combat cellular stress and protect neighboring neurons from injury. In order to fulfill this important role, astrocytes must sense and respond to toxic stimuli, perhaps including stimuli that are severely stressful and kill some of the astrocytes. The present study demonstrates that primary astrocytes that managed to survive severe proteotoxic stress were protected against subsequent challenges. These findings suggest that the phenomenon of preconditioning or tolerance can be extended from mild to severe stress for this cell type. Astrocytic stress adaptation lasted at least 96 h, the longest interval tested. Heat shock protein 70 (Hsp70) was raised in stressed astrocytes, but inhibition of neither Hsp70 nor Hsp32 activity abolished their resistance against a second proteotoxic challenge. Only inhibition of glutathione synthesis abolished astrocytic stress adaptation, consistent with our previous report. Primary neurons were plated upon previously stressed astrocytes, and the cocultures were then exposed to another proteotoxic challenge. Severely stressed astrocytes were still able to protect neighboring neurons against this injury, and the protection was unexpectedly independent of glutathione synthesis. Stressed astrocytes were even able to protect neurons after simultaneous application of proteasome and Hsp70 inhibitors, which otherwise elicited synergistic, severe loss of neurons when applied together. Astrocyte-induced neuroprotection against proteotoxicity was not elicited with astrocyte-conditioned media, suggesting that physical cell-to-cell contacts may be essential. These findings suggest that astrocytes may adapt to severe stress so that they can continue to protect neighboring cell types from profound injury.

Performance evaluation of the Helena V8 capillary electrophoresis system.

OBJECTIVES: To compare the performance characteristics of the Helena V8® and Sebia CAPILLARYS2® automated capillary electrophoresis systems to agarose gel serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE) using the Helena SPIFE3000®. DESIGN AND METHODS: Serum protein electrophoresis and immunosubtraction was performed on 100 consecutive patient samples comparing two capillary-electrophoresis platforms with agarose-gel SPE and IFE; IFE was used as the gold standard. Chart review was performed on patients where results were discordant between methods. Analytical precision was determined using Sebia's normal and abnormal controls. RESULTS: The sensitivities of the CAPILLARYS2, V8, and SPIFE3000 agarose gel for identification of monoclonal gammopathies were respectively 97.4 (95%CI 91.1-100), 92.3 (95%CI 82.2-100), and 89.9 (95%CI 79.1-97.6). The specificities of the CAPILLARYS2, V8, and SPIFE3000 agarose gel were respectively 57.6 (95%CI 45.0-70.2), 72.2 (95%CI 61.0-83.3), and 75.4 (95%CI 60-82.8). These analytical performance characteristics were statistically equivalent between systems (P>0.05). The analytical precision of the capillary-based methods was also statistically equivalent. Chart review of available data from discordant samples revealed that 7/10 patients had a history of multiple myeloma or known monoclonal gammopathy and were being treated or monitored. All discordant samples had low concentration monoclonal proteins (<0.3g/dL). Both capillary-based methods performed poorly (collectively <50% accuracy) at detecting low concentration non-IgG antibodies (IgA, IgM, and light chain monoclonal gammopathies) compared to IFE. CONCLUSIONS: The Helena V8 and Sebia CAPILLARYS2 were analytically equivalent to the SIFE3000 for identification of IgG monoclonal gammopathies >0.3g/dL. Interpreters using the automated immunotyping/immunosubstraction systems performed poorly at detecting low concentration and non-IgG monoclonal gammopathies.

Distinct pathways mediate the sorting of tail-anchored proteins to the plastid outer envelope.

BACKGROUND: Tail-anchored (TA) proteins are a distinct class of membrane proteins that are sorted post-translationally to various organelles and function in a number of important cellular processes, including redox reactions, vesicular trafficking and protein translocation. While the molecular targeting signals and pathways responsible for sorting TA proteins to their correct intracellular destinations in yeasts and mammals have begun to be characterized, relatively little is known about TA protein biogenesis in plant cells, especially for those sorted to the plastid outer envelope. METHODOLOGY/PRINCIPAL FINDINGS: Here we investigated the biogenesis of three plastid TA proteins, including the 33-kDa and 34-kDa GTPases of the translocon at the outer envelope of chloroplasts (Toc33 and Toc34) and a novel 9-kDa protein of unknown function that we define here as an outer envelope TA protein (OEP9). Using a combination of in vivo and in vitro assays we show that OEP9 utilizes a different sorting pathway than that used by Toc33 and Toc34. For instance, while all three TA proteins interact with the cytosolic OEP chaperone/receptor, AKR2A, the plastid targeting information within OEP9 is distinct from that within Toc33 and Toc34. Toc33 and Toc34 also appear to differ from OEP9 in that their insertion is dependent on themselves and the unique lipid composition of the plastid outer envelope. By contrast, the insertion of OEP9 into the plastid outer envelope occurs in a proteinaceous-dependent, but Toc33/34-independent manner and membrane lipids appear to serve primarily to facilitate normal thermodynamic integration of this TA protein. CONCLUSIONS/SIGNIFICANCE: Collectively, the results provide evidence in support of at least two sorting pathways for plastid TA outer envelope proteins and shed light on not only the complex diversity of pathways involved in the targeting and insertion of proteins into plastids, but also the molecular mechanisms that underlie the delivery of TA proteins to their proper intracellular locations in general.

Cell-free analysis of tail-anchor protein targeting to membranes.

We describe cell-free strategies for analysis of the biogenesis of membrane proteins. Special emphasis is placed on the unique challenges presented by tail-anchor proteins for the analysis of: targeting specificity, binding to membranes such as endoplasmic reticulum, mitochondria and pure lipid membranes, protein topology in the membrane and the dependence of various steps on additional proteins. Tail-anchor proteins such as cytochrome b(5), Bcl-2 and Bcl-X(L) are used to address the suitability of both cell-free techniques based on in vitro transcription translation systems and the use of purified recombinant protein and pure lipid membranes. Together these cell-free systems permit detailed characterization of membrane protein biogenesis.

Novel targeting signals mediate the sorting of different isoforms of the tail-anchored membrane protein cytochrome b5 to either endoplasmic reticulum or mitochondria.

Tail-anchored membrane proteins are a class of proteins that are targeted posttranslationally to various organelles and integrated by a single segment of hydrophobic amino acids located near the C terminus. Although the localization of tail-anchored proteins in specific subcellular compartments in plant cells is essential for their biological function, the molecular targeting signals responsible for sorting these proteins are not well defined. Here, we describe the biogenesis of four closely related tung (Aleurites fordii) cytochrome b5 isoforms (Cb5-A, -B, -C, and -D), which are small tail-anchored proteins that play an essential role in many cellular processes, including lipid biosynthesis. Using a combination of in vivo and in vitro assays, we show that Cb5-A, -B, and -C are targeted exclusively to the endoplasmic reticulum (ER), whereas Cb5-D is targeted specifically to mitochondrial outer membranes. Comprehensive mutational analyses of ER and mitochondrial Cb5s revealed that their C termini, including transmembrane domains (TMD) and tail regions, contained several unique physicochemical and sequence-specific characteristics that defined organelle-specific targeting motifs. Mitochondrial targeting of Cb5 was mediated by a combination of hydrophilic amino acids along one face of the TMD, an enrichment of branched beta-carbon-containing residues in the medial portion of the TMD, and a dibasic -R-R/K/H-x motif in the C-terminal tail. By contrast, ER targeting of Cb5 depended primarily upon the overall length and hydrophobicity of the TMD, although an -R/H-x-Y/F- motif in the tail was also a targeting determinant. Collectively, the results presented provide significant insight into the early biogenetic events required for entry of tail-anchored proteins into either the ER or mitochondrial targeting pathways.