Ultrafast intraoperative parathyroid hormone monitoring system: prospective, multicentre, clinical validity study.

BACKGROUND: Intraoperative parathyroid hormone (PTH) monitoring is a proven and reliable adjunct to parathyroid surgery, able to improve the outcomes and efficiency of the diagnostic and therapeutic pathway for patients with primary hyperparathyroidism. This study evaluated the innovative, compact, fully automated NBCL CONNECT Analyzer, which can measure whole-blood PTH in 5 min. METHODS: A prospective multicentre study was conducted in stages: results reviews, recommendations, and implementation of improvements to the mechanical design, components of cartridges, calibration, and sampling protocols. Patients undergoing parathyroidectomy had PTH levels measured on the Analyzer and main laboratory platforms, either Roche or Abbott. The Miami criterion of a 50% drop in PTH concentration was used to define biochemical cure during surgery, and normal postoperative calcium level as cure of primary hyperparathyroidism. Measurements on the Analyzer were done by laboratory staff in London and nurses in Stuttgart. The Pearson coefficient (R) and Wilcoxon test were used for statistical analysis. RESULTS: Some 234 patients (55 male, 179 female) with a median age of 58.5 (age full range 15-88) years underwent parathyroidectomy (195 minimally invasive, 38 bilateral neck exploration, 1 thoracoscopic; 12 conversions) for primary hyperparathyroidism between November 2021 and July 2022. Primary hyperparathyroidism was cured in 225 patients (96.2%). The sensitivity, specificity, and overall accuracy of the Analyzer assay in predicting biochemical cure were 83.9, 100, and 84.8% in phase 1; 91.2, 100, and 91.3% in phase 2; and 98.6, 100, and 98.6% in phase 3. There were no false-positive results (positive predictive value 100%). Correlations between Analyzer measurements and those obtained using the Roche device were very strong (R = 0.98, P < 0.001 in phase 1; R = 0.92, P < 0.001 in phase 2; R = 0.94, P < 0.001 in phase 3), and correlations for Analyzer readings versus those from the Abbott platform were strong (R = 0.82, P < 0.001; R = 0.89, P < 0.001; R = 0.91, P < 0.001). The Analyzer showed continued good mechanical performance, with stable and repeatable operations (calibrations, quality controls). Introducing a stricter sampling protocol and improvements in the clot-detecting system led to a decrease in the number of clotted samples and false-negative results. Outcomes were not affected by measurements performed either by nurses or laboratory staff. CONCLUSION: Intraoperative PTH monitoring during parathyroid surgery can be done accurately, simply, and quickly in whole blood using the Analyzer.

N-acetylation and phosphorylation of Sec complex subunits in the ER membrane.

BACKGROUND: Covalent modifications of proteins provide a mechanism to control protein function. Here, we have investigated modifications of the heptameric Sec complex which is responsible for post-translational protein import into the endoplasmic reticulum (ER). It consists of the Sec61 complex (Sec61p, Sbh1p, Sss1p) which on its own mediates cotranslational protein import into the ER and the Sec63 complex (Sec63p, Sec62p, Sec71p, Sec72p). Little is known about the biogenesis and regulation of individual Sec complex subunits. RESULTS: We show that Sbh1p when it is part of the Sec61 complex is phosphorylated on T5 which is flanked by proline residues. The phosphorylation site is conserved in mammalian Sec61ß, but only partially in birds, and not in other vertebrates or unicellular eukaryotes, suggesting convergent evolution. Mutation of T5 to A did not affect the ability of mutant Sbh1p to complement the growth defect in a Δsbh1Δsbh2 strain, and did not result in a hypophosphorylated protein which shows that alternate sites can be used by the T5 kinase. A survey of yeast phosphoproteome data shows that Sbh1p can be phosphorylated on multiple sites which are organized in two patches, one at the N-terminus of its cytosolic domain, the other proximal to the transmembrane domain. Surprisingly, although N-acetylation has been shown to interfere with ER targeting, we found that both Sbh1p and Sec62p are cotranslationally N-acetylated by NatA, and N-acetyl-proteome data indicate that Sec61p is modified by the same enzyme. Mutation of the N-acetylation site, however, did not affect Sec62p function in posttranslational protein import into the ER. Disabling NatA resulted in growth retardation, but not in co- or posttranslational translocation defects or instability of Sec62p or Sbh1p. CONCLUSIONS: We conclude that N-acetylation of transmembrane and tail-anchored proteins does not interfere with their ER-targeting, and that Sbh1p phosphorylation on T5, which is not present in Sbh2p, plays a non-essential role specific to the Sec61 complex.

A human genome-wide screen for regulators of clathrin-coated vesicle formation reveals an unexpected role for the V-ATPase.

Clathrin-mediated endocytosis is essential for a wide range of cellular functions. We used a multi-step siRNA-based screening strategy to identify regulators of the first step in clathrin-mediated endocytosis, formation of clathrin-coated vesicles (CCVs) at the plasma membrane. A primary genome-wide screen identified 334 hits that caused accumulation of CCV cargo on the cell surface. A secondary screen identified 92 hits that inhibited cargo uptake and/or altered the morphology of clathrin-coated structures. The hits include components of four functional complexes: coat proteins, V-ATPase subunits, spliceosome-associated proteins and acetyltransferase subunits. Electron microscopy revealed that V-ATPase depletion caused the cell to form aberrant non-constricted clathrin-coated structures at the plasma membrane. The V-ATPase-knockdown phenotype was rescued by addition of exogenous cholesterol, indicating that the knockdown blocks clathrin-mediated endocytosis by preventing cholesterol from recycling from endosomes back to the plasma membrane.

Association of serum sex steroid receptor bioactivity and sex steroid hormones with breast cancer risk in postmenopausal women.

Postmenopausal women with elevated serum sex steroids have an increased risk of breast cancer. Most of this risk is believed to be exerted through binding of the sex steroids to their receptors. For the first time, we investigate the association of estrogen receptor (ER) and androgen receptor (AR) serum bioactivity (SB) in addition to hormone levels in samples from women with breast cancer collected before diagnosis. Two hundred postmenopausal women participating in the UK Collaborative Trial of Ovarian Cancer Screening who developed ER-positive breast cancer 0.6-5 years after sample donation were identified and matched to 400 controls. ER and AR bioassays were used to measure ERα, ERß, and AR SB. Androgen and estrogen levels were measured with immunoassays. Subjects were classified according to quintiles of the respective marker among controls and the associations between SB and hormones with breast cancer risk were determined by logistic regression analysis. ERα and ERß SB were significantly higher before diagnosis compared with controls, while estrogens showed no difference. Women had a twofold increased breast cancer risk if ERα SB (odds ratio (OR), 2.114; 95% confidence interval (CI), 1.050-4.425; P=0.040) was in the top quintile >2 years before diagnosis or estrone (OR, 2.205; 95% CI, 1.104-4.586; P=0.029) was in the top quintile <2 years before diagnosis. AR showed no significant association with breast cancer while androstenedione (OR, 3.187; 95% CI, 1.738-6.044; P=0.0003) and testosterone (OR, 2.145; 95% CI, 1.256-3.712; P=0.006) were significantly higher compared with controls and showed a strong association with an almost threefold increased breast cancer risk independent of time to diagnosis. This study provides further evidence on the association of androgens and estrogens with breast cancer. In addition, it reports that high ER but not AR SB is associated with increased breast risk >2 years before diagnosis.

The transmembrane domain is sufficient for Sbh1p function, its association with the Sec61 complex, and interaction with Rtn1p.

The Sec61 protein translocation complex in the endoplasmic reticulum (ER) membrane is composed of three subunits. The alpha-subunit, called Sec61p in yeast, is a multispanning membrane protein that forms the protein conducting channel. The functions of the smaller, carboxyl-terminally tail-anchored beta subunit Sbh1p, its close homologue Sbh2p, and the gamma subunit Sss1p are not well understood. Here we show that co-translational protein translocation into the ER is reduced in sbh1Delta sbh2Delta cells, whereas there is a limited reduction of post-translational translocation and no effect on export of a mutant form of alpha-factor precursor for ER-associated degradation in the cytosol. The translocation defect and the temperature-sensitive growth phenotype of sbh1Delta sbh2Delta cells were rescued by expression of the transmembrane domain of Sbh1p alone, and the Sbh1p transmembrane domain was sufficient for coimmunoprecipitation with Sec61p and Sss1p. Furthermore, we show that Sbh1p co-precipitates with the ER transmembrane protein Rtn1p. Sbh1p-Rtn1p complexes do not appear to contain Sss1p and Sec61p. Our results define the transmembrane domain as the minimal functional domain of the Sec61beta homologue Sbh1p in ER translocation, identify a novel interaction partner for Shb1p, and imply that Sbh1p has additional functions that are not directly linked to protein translocation in association with the Sec61 complex.