Improved Neutron Lifetime Measurement with UCNτ.

We report an improved measurement of the free neutron lifetime τ_{n} using the UCNτ apparatus at the Los Alamos Neutron Science Center. We count a total of approximately 38×10^{6} surviving ultracold neutrons (UCNs) after storing in UCNτ's magnetogravitational trap over two data acquisition campaigns in 2017 and 2018. We extract τ_{n} from three blinded, independent analyses by both pairing long and short storage time runs to find a set of replicate τ_{n} measurements and by performing a global likelihood fit to all data while self-consistently incorporating the ß-decay lifetime. Both techniques achieve consistent results and find a value τ_{n}=877.75±0.28_{stat}+0.22/-0.16_{syst} s. With this sensitivity, neutron lifetime experiments now directly address the impact of recent refinements in our understanding of the standard model for neutron decay.

Search for the Neutron Decay n→X+γ, Where X is a Dark Matter Particle.

Fornal and Grinstein recently proposed that the discrepancy between two different methods of neutron lifetime measurements, the beam and bottle methods, can be explained by a previously unobserved dark matter decay mode, n→X+γ. We perform a search for this decay mode over the allowed range of energies of the monoenergetic γ ray for X to be dark matter. A Compton-suppressed high-purity germanium detector is used to identify γ rays from neutron decay in a nickel-phosphorous-coated stainless-steel bottle. A combination of Monte Carlo and radioactive source calibrations is used to determine the absolute efficiency for detecting γ rays arising from the dark matter decay mode. We exclude the possibility of a sufficiently strong branch to explain the lifetime discrepancy with 97% confidence.

Integrated surveillance of human respiratory viruses in addition to SARS-CoV-2 in a public testing facility in the Netherlands.

BACKGROUND: SARS-CoV-2 prevention measures impact the circulation of other respiratory viruses. Surveillance in the network of general practitioners is hampered by widespread testing for SARS-CoV-2 in public testing facilities. OBJECTIVES: To evaluate integrated community surveillance of SARS-CoV-2 and other respiratory viruses and describe epidemiological trends. STUDY DESIGN: Respiratory surveillance was set up within an existing SARS-CoV-2 public testing facility. Community-dwelling (a)symptomatic persons provided consent for completion of a questionnaire and additional testing on residual material from swabs taken for SARS-CoV-2 RT-PCR (Allplex Seegene). Daily, a random subset was tested for sixteen respiratory viruses by multiplex realtime PCRs (Seegene). RESULTS: Between October 6th (week 40) 2021 and April 22nd (week 16) 2022, 3,969 subjects were tested. The weekly median age ranged from 23 to 39 years. The prevalence of respiratory symptoms ranged from 98.5% (week 40) to 27.4% (week 1). The prevalence of detection of any respiratory virus (including SARS-CoV-2), ranged from 19.6% in week 49 to 75.3% in week 14. SARS-CoV-2 prevalence ranged from 2.2% (week 40) to 63.3% (week 14). Overall, SARS-CoV-2 was detected most frequently (27.3%), followed by rhinoviruses (14.6%, range 3.5-47.8%) and seasonal coronaviruses (3.7%, range 0-10.4%, mostly 229E and OC43). Influenzavirus was detected in 3.0% of participants from week 6 onwards. CONCLUSIONS: Integrated respiratory viral surveillance within public testing facilities is feasible and informative. Prevalences may be affected by changes in SARS-CoV-2 prevention and testing policies. Population characteristics help to interpret trends over time. Integrated surveillance may inform policymakers and hospitals for adequate response measures during respiratory seasons.

Proteolytic cleavage of haptoglobin occurs in a subcompartment of the endoplasmic reticulum: evidence from membrane fusion in vitro.

The primary translation product of haptoglobin mRNA is a 45-kD polypeptide which is proteolytically cleaved shortly after its synthesis. Previous studies have indicated that the cleavage of this proform of haptoglobin occurs in the ER. In an attempt to characterize the cleaving enzyme, we found that upon incubation of microsomes from rat hepatocytes pulse labeled with [35S]methionine, little cleavage of labeled prohaptoglobin occurred. In contrast, when cells whose cytoplasmic proteins had been released by saponin treatment were incubated, 30-40% of the prohaptoglobin was cleaved. The addition of GTP caused a twofold stimulation, which was abolished by the nonhydrolyzable analog GTP gamma S. With a homogenate of the cells, the addition of GTP resulted in a fourfold stimulation of the degree of cleavage--from 15 to 60%. Differential centrifugation revealed that most of the cleaving activity resided in membranes sedimenting similarly to mitochondria and to a small fraction of the ER. These rapidly sedimenting membranes were therefore prepared from a rat liver homogenate. Upon treatment with high salt, light membranes were released which, when incubated with microsomes of pulse-labeled hepatocytes in the presence of detergent (and in the absence of GTP), induced specific cleavage of prohaptoglobin. The cleaving enzyme had an alkaline pH optimum indicating that it was not of lysosomal origin. These results suggest that cleavage of prohaptoglobin occurs in a subcompartment of the ER. Apparently, the connection between this compartment and the bulk of the ER is broken upon saponin treatment or homogenization but can be reestablished through a process requiring GTP hydrolysis.

Transient activity of Golgi-like membranes as donors of vesicular stomatitis viral glycoprotein in vitro.

Previous reports demonstrated that the vesicular stomatitis viral glycoprotein (G protein), initially present in membranes of a Chinese hamster ovary mutant cell line (clone 15B) that is incapable of terminal glycosylation, can be transferred in vitro to exogenous Golgi membranes and there glycosylated (E. Fries and J. E. Rothman, 1980, Proc. Natl. Acad. Sci. U. S. A. 77:3870-3874; and J. E. Rothman and E. Fries, 1981, J. Cell Biol. 89:162-168). Here we present evidence that Golgi-like membranes serve as donors of G protein in this process. Pulse-chase experiments revealed that the donor activity of membranes is greatest at approximately 10 min of chase, a time when G protein has been shown to have arrived in Golgi stacks (J. E. Bergmann, K. T. Tokuyasu, and S. J. Singer, 1981, Proc. Natl. Acad. Sci. U. S. A. 78:1746-1750). Additional evidence that the G protein that is transferred to exogenous Golgi membranes in vitro had already entered the Golgi membranes in vivo was provided by observations that its oligosaccharides had already been trimmed, and that its distribution in a sucrose density gradient was coincident with that of enzymatic markers of Golgi membranes. The capacity of this Golgi-like membrane to serve as donor is transient, declining within 5 min after "trimming" in vivo as the G protein enters a "nontransferable" pool. The rapidity of the process suggests that both the "transferable" and "nontransferable" pools of G protein reside in Golgi-like membranes.

Transport of newly synthesized vesicular stomatitis viral glycoprotein to purified Golgi membranes.

In a previous communication we reported that the newly synthesized membrane glycoprotein of vesicular stomatitis virus could be transported in crude extracts of CHO cells from endoplasmic reticulum-derived membranes to membranes of the Golgi complex. This conclusion was an indirect one, based on the terminal glycosylation of this glycoprotein, a reaction that was dependent upon a Golgi-specific enzyme, UDP-GlcNAc transferase I. We show here that the Golgi fraction of rat liver will substitute for members of CHO cells as a source of transferase I in this reaction. The use of highly purified fractions of liver Golgi membranes, coupled with the ability to recover these membranes from incubations, has now permitted a direct demonstration of net transport of G protein to these heterologous Golgi membranes. This transport reaction is specific, in that the smooth endoplasmic reticulum fraction will not substitute for the Golgi fraction, is quantitatively significant, involving at least 30% of the viral glycoprotein, and is sustained only in the presence of both ATP and a soluble, cytosol fraction of liver cells.

Reconstitution of Semliki forest virus membrane.

The spike glycoproteins of the Semliki forest virus membrane have been incorporated into vesicular phospholipid bilayers by a detergent-dialysis method. The detergent used was beta-D-octylglucoside which is nonionic and has an exceptionally high critical micellar concentration which facilitates rapid removal by dialysis. The vesicles obtained were of varying sizes and had spikes on their surface. Two classes of vesicles were preferentially formed, small protein-rich and large lipid-rich (average lipid to protein weight ratios, 0.22 and 3.5, respectively). Both classes of vesicles retained the hemagglutinating activity of the virus. The proteins were attached to the lipid bilayer by hydrophobic peptide segments, as in the viral membrane. Most of the proteins were accessible to proteolytic digestion from the outside, suggesting an asymmetric orientation.

On the entry of Semliki forest virus into BHK-21 cells.

The pathway by which semliki forest virus (SFV), a membrane-containing animal virus, enters BHK-21 cells was studied morphologically and biochemically. After attaching to the cell surface, the majority of viruses was rapidly trapped into coated pits, internalized by endocytosis in coated vesicles, and sequestered into intracellular vacuoles and lysosomes. Direct penetration of viruses through the plasma membrane was never observed. To assess the possible involvement of lysosomes in the release of the genome into the cytoplasm, the effect of five lysosomotropic agents, known to increase the lysosomal pH, was tested. All of these agents inhibited SFV infectivity and one, chloroquine (the agent studied in most detail), inhibited a very early step in the infection but had no effect on binding, endocytosis, or intracellular distribution of SFV. Thus, the inhibitory effect was concluded to be either on penetration of the nucelocapsid into the cytoplasm or on uncoating of the viral RNA. Possible mechanisms for the penetration of the genome into the cytoplasm were studied in vitro, using phospholipids-cholesterol liposomes and isolated SFV. When the pH was 6.0 or lower, efficient fusion of the viral membranes and the liposomal membranes occurred, resulting in the transfer of the nucleocapsid into the liposomes. Infection of cells could also be induced by brief low pH treatment of cells with bound SFV under conditions where the normal infection route was blocked. The results suggest that the penetration of the viral genome into the cytosol takes place intracellularly through fusion between the limiting membrane of intracellular vacuoles and the membrane of viruses contained within them. The low pH required for fusion together with the inhibitory effect of lysosomotropic agents implicate lysosomes, or other intracellular vacuoles with sufficiently low pH, as the main sites of penetration.

Differential arrest of secretory protein transport in cultured rat hepatocytes by azide treatment.

The effect of reduced cellular ATP content on intracellular transport of two secretory proteins, albumin and haptoglobin, in isolated rat hepatocytes was studied. The cells were labeled with [35S]methionine and the cellular ATP content was then rapidly reduced to different stable levels by incubation with azide at different concentrations (2.0-10 mM). The amount of the radioactively labeled secretory proteins in the cells and in the medium after 150 min of incubation was determined by immunoprecipitation followed by gel electrophoresis, fluorography, and densitometry. At progressively lower ATP levels, down to 50% of normal, the protein secretion was unaffected, whereas at even lower levels an increasing portion of the proteins remained in the cells; at 30 and 10% of normal ATP level, 25 and 75% of albumin, respectively, was arrested intracellularly. Analysis of the carbohydrate structure of intracellularly arrested haptoglobin showed that in cells with an ATP level of approximately 30% of normal, the majority of haptoglobin molecules (55%) were fully or partially resistant to endoglycosidase H. This result indicates that exit from the medial and/or the trans part of the Golgi complex (GC) was inhibited under these conditions. It also shows that the protein had accumulated in the GC, since under normal conditions the fraction of the intracellular haptoglobin that is endoglycosidase H resistant is approximately 10%. By similar criteria it was found that at ATP levels below 10% of normal transport of haptoglobin from the endoplasmic reticulum to the medial GC (and possibly also to the cis GC) as well as from the trans GC to the medium were blocked.

Several daily measurements are necessary to reliably assess the cortisol rise after awakening: state- and trait components.

The cortisol rise after awakening (CAR) is a frequently applied measure of pituitary-adrenal activity. This measure seems to reflect the acrophase of the diurnal cycle and can easily be assessed in saliva samples, collected by the proband or patient under real life conditions. Since different state and trait factors affect the CAR, we here address the questions (a) to which extent state and trait factors affect the CAR, and (b) how often cortisol measures after awakening have to be taken to obtain reliable results. In this study, we assessed the CAR on 6 consecutive days. After applying structural equation models and correlation analyses, we conclude that (a) the CAR of a single day is determined to a great extent by situational factors and only for a small proportion by trait factors and (b) from two (AUC(t)) to six (AUC(i)) days are necessary to achieve reliable trait measures, since state factors bias data from a single day.

Evaluation of the analytical performance of the new Abbott RealTime RT-PCRs for the quantitative detection of HCV and HIV-1 RNA.

BACKGROUND: Despite FDA approval and CE marking of commercial tests, manufacturer independent testing of technical aspects is important. OBJECTIVES: To evaluate the analytical performance of the new Abbott RealTime HCV and HIV-1 viral load tests. STUDY DESIGN: Sensitivity, specificity and inter-/intra-assay variation were investigated. The HCV and HIV-1 assays were compared with Siemens bDNA 3.0 and Roche Cobas Monitor 2.0, respectively, on diagnostic samples. RESULTS: Lower isolation volumes on the M1000 gave minor but statistically significant lower quantitative values. Minor differences were observed in the lower limit of detection relative to the specification given by the manufacturer. Inter-/intra-assay coefficients of variations ranged from 0.31 to 4.75 between 5.0 x 10(4) and 5.0 x 10(2) copies/mL. Both the HCV and HIV-1 Abbott RealTime tests did not show a geno-/sub-type dependent under-quantification on WHO reference panels, quality control panels or clinical specimens. The Abbott RealTime HIV-1 viral load assay detected subtype O whereas several other systems failed to detect this subtype. CONCLUSION: The technical aspects of the HCV and HIV-1 RealTime viral load assays on the M2000 system make it attractive for use in routine diagnostic settings.

Racial misclassification of Native Americans in a surveillance, epidemiology, and end results cancer registry.

BACKGROUND: The cancer incidence for all sites has been reported to be lower in Native Americans than in White Americans. Concerns have been expressed, however, that the observed low incidence may be a result of inaccurate reporting of race. PURPOSE: The objective of this study was to investigate the extent to which racial misclassification may contribute to the observed low cancer incidence among Native Americans. METHODS: A registry of individuals eligible to receive medical services funded by the Indian Health Service was linked by computer to the Puget Sound Surveillance, Epidemiology, and End Results (SEER) cancer registry. RESULTS: Only 137 (60%) of the patients with invasive cancer registered with the Indian Health Service and for whom race was recorded were identified as Native Americans in the SEER registry. Similarly, 55 (69%) of 80 in situ cervical cancer case patients were classified as Native American. A strong association was observed between Native-American blood quantum level and racial misclassification. CONCLUSION: The results of this study indicate that the observed low cancer incidence in Native Americans relative to Whites in the northwest United States is at least partially attributable to racial misclassification in the SEER cancer registry.

Determination of Triton X-100 binding to membrane proteins by polyacrylamide gel electrophoresis.

The molecular weight of proteins in protein-detergent complexes can be determined from ultracentrifugation experiments if the amount of bound detergent is known. A new sensitive method to measure the binding of the nonionic detergent Triton X-100 to proteins has been developed. For the membrane proteins studied, less than 50 mug of protein was required to achieve an accuracy of 10% in the determination of the detergent-protein weight ratio. The proteins were equilibrated with the detergent by electrophoresis into polyacrylamide gels containing radioactively labelled Triton X-100. The gels were then sliced and the amount of bound detergent calculated from the increase in radio-activity in the slices containing the protein zone. The amounts of protein were determined by amino acid analysis of identical protein zones cut from gels running parallel.

Solubilization of the Semliki Forest virus membrane with sodium deoxycholate.

The effects of increasing concentrations of sodium deoxycholate on Semliki Forest have been studied. Sodium deoxycholate begins to bind to the virus at less than 0.1 mM free equilibrium concentration and causes lysis of the viral membrane at 0.9 +/- 0.1 mM free equilibrium concentration when 2.2 +/- 0.2 - 103 mol of sodium deoxycholate are bound per mol of virus. Liberation of proteins from the membrane begins at 1.5 +/- 0.1 mM sodium deoxycholate and the proteins released are virtually free from phospholipid above 2.0 mM sodium deoxycholate. The overall mechanism of sodium deoxycholate solubilization of the viral membrane resembles that of Triton X-100 and sodium dodecyl sulphate except that with sodium deoxycholate the various stages of membrane disruption occur at about 10-fold higher equilibrium free detergent concentrations. At sodium deoxycholate concentrations higher than 2.3 mM the viral spike glycoproteins can be separated by sucrose gradient centrifugation or gel filtration into constituent polypeptides E1, E2 and E3. E1 carries the haemagglutinating activity of the virus.

Increase of bikunin and alpha1-microglobulin concentrations in urine of rats during pregnancy is due to decreased tubular reabsorption.

Bikunin and alpha1-microglobulin are two plasma proteins of about 25 kDa which are made in the liver from a common precursor. The concentration of bikunin in human urine has been shown to increase several fold during various conditions of stress. The mechanism behind this increase is unknown. We have studied pregnant rats and found that the bikunin and alpha1-microglobulin levels in their urine increased 3-fold towards the end of the pregnancy, whereas those of albumin and orosomucoid did not. There were no significant changes in either the bikunin/alpha1-microglobulin mRNA level or the concentrations of the two proteins in serum. These findings imply that the synthesis and the clearance rates of bikunin and alpha1-microglobulin are normal during pregnancy but that the tubular reabsorption of these proteins is decreased.

Permeabilization of hepatocytes by a saponin and the effects of dextran.

The process by which a saponin derived from Gypsophila plants permeabilizes rat hepatocytes was studied. When monolayer cultures were incubated with 25 micrograms/ml saponin in phosphate buffered saline, the amount of cell-bound saponin increased for at least 90 min. Release of intracellular K+ started immediately, with a t1/2 of about 5 min. ATP and lactate dehydrogenase (LDH) began to appear in the medium only after lag periods of 10 to 20 min with t1/2s of 20 to 30 min. Removing the saponin from the medium after 15 min stopped any further release of ATP and LDH, showing that increased permeability to small ions alone does not lead to lysis by colloid osmotic pressure. However, the lysis that occurred upon 30 min continuous incubation with the saponin could be inhibited (delayed) by the addition of an osmotically active compound - a dextran. These results indicate that increasing binding of the saponin destabilizes the plasma membrane so that it will rupture from the colloid osmotic pressure.

Bikunin--not just a plasma proteinase inhibitor.

Bikunin is a plasma proteinase inhibitor that has received little attention in the past, probably because its activity towards various proteinases was found to be relatively weak in early work. It was recently discovered, however, that bikunin effectively inhibits a proteinase that seems to be involved in the metastasis of tumour cells--cell surface plasmin--and that a fragment of bikunin inhibits two proteinases of the coagulation pathway--factor Xa and kallikrein. Furthermore, it has been found that bikunin has other properties, such as the ability to modulate cell growth and to block cellular calcium uptake. Most of the bikunin in the blood occurs as a covalently linked subunit of the proteins pre- and inter-alpha-inhibitor. In this form bikunin lacks some of its known activities, and there is evidence that its release by partial proteolytic degradation may function as a regulatory mechanism. Although the physiological function of bikunin still remains to be established, current data suggest that this protein plays a role in inflammation. Further studies could therefore lead to results of therapeutical value.

Synthesis of alpha 1-microglobulin in cultured rat hepatocytes is stimulated by interleukin-6, leukemia inhibitory factor, dexamethasone and retinoic acid.

The secretion of alpha 1-microglobulin by primary cultures of rat hepatocytes was found to increase upon the addition of interleukin-6 or leukemia inhibitory factor, two mediators of acute phase response. This stimulatory effect was further enhanced by dexamethasone. alpha 1-Microglobulin is synthesized as a precursor also containing bikunin, and the precursor protein is cleaved shortly before secretion. Our results therefore suggest that both alpha 1-microglobulin and bikunin are acute phase reactants in rat hepatocytes. Furthermore, we found that retinoic acid, previously shown to be involved in the regulation of cell differentiation and development, also stimulated alpha 1-microglobulin synthesis. Only free, uncomplexed alpha 1-microglobulin (28,000 Da) was detected in the hepatocyte media, suggesting that the complex between alpha 1-microglobulin and alpha 1-inhibitor 3, found in rat serum, is formed outside the hepatocyte.

Widely used, commercially available Chlamydia pneumoniae antigen contaminated with mycoplasma.

Mycoplasma contamination was detected in a widely used commercially available Chlamydia pneumoniae antigen preparation. Contamination was studied with a mycoplasma group-specific 16S rRNA polymerase chain reaction (PCR) and sequence analysis. Several lots of the purified C. pneumoniae antigen from the Washington Research Foundation appeared to be contaminated with the same Mycoplasma species, which appeared to be closely related to M. arginini. Antigen slides prepared for the detection of chlamydia antibodies by MRL Diagnostics were contaminated with the same Mycoplasma sp. Chlamydia antigen slides from Labsystems OY and two chlamydia complement fixation reagents (Virion International Distribution Ltd and Behring Werke) were not contaminated. It is concluded that commercially available C. pneumoniae antigens may contain mycoplasma antigens as well. Although the impact of such mycoplasma contamination on the results of chlamydia serology may not be significant, routine screening of all antigen preparations obtained by tissue culture before their distribution and use is recommended.

Binding of Zn(2+) to the plasma protein inter-alpha-inhibitor.

Inter-alpha-inhibitor (IalphaI) is a serum protein consisting of a chondroitin-sulfate-containing protein of 25 kDa (bikunin) and two other polypeptides of 75-80 kDa (heavy chains 1 and 2). The physiological function of IalphaI is unclear but recent results suggest that it is required for the formation of the extracellular matrix of certain cell types and that it has anti-inflammatory activity. It was previously reported that IalphaI isolated from serum contains bound Zn(2+), but details of this binding are lacking. Using equilibrium dialysis, we have found that when the free Zn(2+) concentration is raised from 0.3 to 50 micromol/L, the number of bound ions increases from 0.1 to 7. The concentration of free Zn(2+) in plasma is in the nanomolar range; our results therefore suggest that inter-alpha-inhibitor does not contain stoichiometric amounts of zinc ions under normal in vivo conditions.