Analytical variation concerning total 25-hydroxyvitamin D measurement, where are we now? A DEQAS review of current assay performance.

The Vitamin D External Quality Assessment Scheme (DEQAS) distributes serum samples globally, on a quarterly basis, to assess participants' performance of specific methods for 25-hydroxyvitamin D (25OHD) and other vitamin D metabolites. In this review an assessment of the state of the art in the performance of 25OHD methods is presented. This assessment is based on an analysis of data submitted by scheme participants for the 2021/22 distribution cycle, which comprised of four distributions each containing five DEQAS samples. These distributions enabled the assessment of performance across a wide concentration range and included samples containing endogenous 25OHD2. Overall analytical performance continues to improve, but there is still significant method variation and bias in some automated methods. These automated methods continue to be challenged in measuring 25OHD at the extremes of the measuring range and in the presence of 25OHD2. LC-MS/MS methods still show superior performance with regards to bias, but are out-performed by some automated methods in terms of assay variability. Through participating in an accuracy based EQA scheme, such as DEQAS, laboratories are able to assess the accuracy of their methods in comparison to a gold standard reference measurement procedure. It is crucial for all laboratories to be aware of the performance and limitations of their 25OHD assays and to educate their users accordingly in order to ensure reliable assessment of vitamin D status.

Spontaneous reversal of the developmental aging of normal human cells following transcriptional reprogramming.

AIM: To determine whether transcriptional reprogramming is capable of reversing the developmental aging of normal human somatic cells to an embryonic state. MATERIALS & METHODS: An isogenic system was utilized to facilitate an accurate assessment of the reprogramming of telomere restriction fragment (TRF) length of aged differentiated cells to that of the human embryonic stem (hES) cell line from which they were originally derived. An hES-derived mortal clonal cell strain EN13 was reprogrammed by SOX2, OCT4 and KLF4. The six resulting induced pluripotent stem (iPS) cell lines were surveyed for telomere length, telomerase activity and telomere-related gene expression. In addition, we measured all these parameters in widely-used hES and iPS cell lines and compared the results to those obtained in the six new isogenic iPS cell lines. RESULTS: We observed variable but relatively long TRF lengths in three widely studied hES cell lines (16.09-21.1 kb) but markedly shorter TRF lengths (6.4-12.6 kb) in five similarly widely studied iPS cell lines. Transcriptome analysis comparing these hES and iPS cell lines showed modest variation in a small subset of genes implicated in telomere length regulation. However, iPS cell lines consistently showed reduced levels of telomerase activity compared with hES cell lines. In order to verify these results in an isogenic background, we generated six iPS cell clones from the hES-derived cell line EN13. These iPS cell clones showed initial telomere lengths comparable to the parental EN13 cells, had telomerase activity, expressed embryonic stem cell markers and had a telomere-related transcriptome similar to hES cells. Subsequent culture of five out of six lines generally showed telomere shortening to lengths similar to that observed in the widely distributed iPS lines. However, the clone EH3, with relatively high levels of telomerase activity, progressively increased TRF length over 60 days of serial culture back to that of the parental hES cell line. CONCLUSION: Prematurely aged (shortened) telomeres appears to be a common feature of iPS cells created by current pluripotency protocols. However, the spontaneous appearance of lines that express sufficient telomerase activity to extend telomere length may allow the reversal of developmental aging in human cells for use in regenerative medicine.

Effect of irrigation with lake water containing microcystins on microcystin content and growth of ryegrass, clover, rape, and lettuce.

The effect of irrigation with lake water containing a variety of microcystins on accumulation of toxins, or toxin metabolites, and plant growth in ryegrass, clover, rape, and lettuce, was investigated in a glasshouse experiment. The plants were grown in sand culture and received either three or six applications of lake water, which was applied either directly to the sand surface or to the plant shoots. As determined by LC-MS, each plant received 170 mug of a mixture of 10 different microcystins per application. Microcystins in plant samples were extracted with 70% methanol and analyzed by Adda-specific ELISA. For the shoot application treatment, microcystins were not present at measurable levels in shoots of ryegrass or rape, but were present in lettuce [0.79 mg/kg dry weight (DW)] and clover (0.20 mg/kg DW). Total microcystin concentration in roots did not vary greatly depending on whether treatment water was applied directly to the sand, or reached the roots via run-off from the shoots. Microcystins in roots were highest in clover (1.45 mg/kg DW), intermediate in lettuce (0.68 mg/kg DW) and low in ryegrass (0.20 mg/kg DW), and rape (0.12 mg/kg DW). There was no evidence for root-to-shoot translocation of microcystins. Three applications of microcystins reduced shoot DW of ryegrass, rape and lettuce, and increased root DW of ryegrass and lettuce. Clover DW was not changed by treatment with microcystins. The results show that irrigation with water containing microcystins has the potential to move microcystins into farm animal and human food chains at concentrations that can exceed recommended tolerable limits.

A cost-effectiveness evaluation of a jail-based chlamydia screening program for men and its impact on their partners in the community.

BACKGROUND: Few cost-effectiveness evaluations of screening men in jails for chlamydia have been published, and none have evaluated the cost-effectiveness of providing partner notification services to the partners of chlamydia-infected inmates. GOAL: The goal of this study was to evaluate the cost-effectiveness of the chlamydia screening and partner notification programs for men conducted by a Massachusetts jail compared with 3 hypothetical alternatives. STUDY DESIGN: Using jail cost and testing data, we used decision analyses to compare the cost and effectiveness of universal screening, age-based screening with 2 age cutoffs, and testing of symptomatic inmates at intake using treated cases of chlamydia and gonorrhea as the primary outcome. We also evaluated the cost-effectiveness of adding partner notification to these alternatives. RESULTS: Universal screening was the most effective and expensive alternative. Age-based screening would have identified slightly fewer cases at half the cost of universal screening. The net cost of partner notification was low. Assuming high sequelae costs in female partners made partner notification a cost-saving intervention. CONCLUSIONS: Age-based screening could lower costs without substantially sacrificing effectiveness. Notifying partners of infected inmates was a cost-effective adjunct to screening inmates.

Control of the cattle louse Bovicola bovis with the fungal pathogen Metarhizium anisopliae.

The effects of the entomopathogenic fungus Metarhizium anisopliae (Metschnikoff) were evaluated against the common louse parasite of cattle, Bovicola bovis (Piaget) (Trichodectidae: Ischnocera). Two different concentrations and formulations of conidial suspensions were applied to contained populations of adult female lice. In vitro, lice immersed in suspensions of M. anisopliae formulated in 0.03% Tween 80 developed infections; at the highest concentration (1x10(8)conidia ml-1) a mean of 71% (+/-11.52%, 95% C.I.) of lice became infected. Lice exposed to the Tween 80 only in vitro, showed high levels of survival and zero infection. In vivo, fungal conidia were applied to louse populations contained in 7 cm diameter circular arenas glued to the backs of Holstein cattle, maintained in controlled climate conditions. Conidia were formulated in either Tween 80 or silicone oil. The treatment with M. anisopliae resulted in high levels of infection and there was no overall difference between the two formulations in the number of infections observed. At the highest concentration (1x10(8)conidia ml-1) a mean of 73% (+/-15.57%, 95% C.I.) lice became infected. It is concluded that the strategic seasonal use of a fungal pathogen on cattle, applied in early winter, may be of value in suppressing the winter increase in abundance, preventing the population increasing to clinically significant levels.

Changes in concentrations of microcystins in rainbow trout, freshwater mussels, and cyanobacteria in Lakes Rotoiti and Rotoehu.

Microcystin concentrations in cyanobacteria and their accumulation in rainbow trout (Oncorhynchus mykiss) and freshwater mussels (Hyridella menziesi) in Lakes Rotoiti and Rotoehu (New Zealand) were investigated. Hatchery rainbow trout were added to an enclosure in Lake Rotoiti where concentrations of microcystins in the phytoplankton and cyanobacterial cell concentrations could be closely monitored. Rainbow trout that were free to roam in the entire area of each lake were also included in the study. Freshwater mussels were suspended subsurface in cages in the enclosure. Phytoplankton samples, rainbow trout liver and muscle tissue, and the tissues of mussels were analyzed for microcystins using the ADDA-ELISA method, and selected samples were analyzed using LC-MS. A maximum concentration of microcystins in the phytoplankton samples of 760 microg L(-1) was recorded in Te Weta Bay, Lake Rotoiti, in March 2004. ELISA results confirmed microcystin immunoreactivity in rainbow trout liver and muscle tissues and in freshwater mussels. The microcystin congeners LR, YR, RR, AR, FR, LA, and WR were detected by LC-MS in caged freshwater mussels in Lake Rotoiti but were not detected in either muscle or liver tissue of rainbow trout. The daily tolerable intake limit of microcystins for human consumption recommended by the World Health Organisation is 0.04 microg kg(-1) day(-1). Modeling was carried out for the human intake of microcystin compounds from rainbow trout muscle tissue, and the potential health risks were estimated, assuming the ADDA-ELISA was determining compounds of toxicity equivalent to microcystin-LR.

A comparison of the insertion characteristics of the laryngeal tube and the laryngeal mask airway: a study of the ED50 propofol requirements.

The purpose of this prospective randomised double-blind study was to determine the effective dose of propofol required for the successful first attempt insertion of the laryngeal tube compared with the laryngeal mask airway in patients co-induced using alfentanil 5 microg.kg(-1), undergoing short elective gynaecological procedures. The first patient in each group received propofol 2.5 mg.kg(-1) for induction. In accordance with Dixon's up-and-down method, the dose of propofol for consecutive patients in each group was varied with increments or decrements of 0.5 mg.kg(-1) based on the previous patient 'all-or-none' purposeful movement response to first attempt of insertion of the randomised device. The ED50 (SD) of propofol was 2.66 (0.86) mg.kg(-1) and 2.33 (0.37) mg.kg(-1) for the laryngeal tube and laryngeal mask patients, respectively, which did not reach statistical significance (p = 0.40). We conclude therefore that the insertion of the two airway devices requires similar bolus doses of propofol when alfentanil is used as the co-induction drug.

p97 and close encounters of every kind: a brief review.

The AAA (ATPase associated with various cellular activities) ATPase, p97, is a hexameric protein of chaperone-like function, which has been reported to interact with a number of proteins of seemingly unrelated functions. For the first time, we report a classification of these proteins and aim to elucidate any common structural or functional features they may share. The interactors are grouped into those containing ubiquitin regulatory X domains, which presumably bind to p97 in the same way as the p47 adaptor, and into non-ubiquitin regulatory X domain proteins of different functional subgroups that may employ a different mode of interaction (assuming they also bind directly to p97 and are not experimental artifacts). Future studies will show whether interacting proteins direct p97 to different cellular pathways or a common one and structural elucidation of these interactions will be crucial in understanding these underlying functions.

Use of ELISA to identify Protoceratium reticulatum as a source of yessotoxin in Norway.

Picked cells of Protoceratium reticulatum collected from five locations in Norway were shown by ELISA analysis to contain yessotoxins (YTXs). The production of yessotoxin (YTX) was verified by culturing followed by LC-MS analysis of one of the Norwegian isolates. This is the first report of the biogenic origin of YTXs in Norway. The sensitivity of the ELISA method made it possible to quantitate YTXs in algal cultures, net-hauls, and in single cells of P. reticulatum. The cells picked from cultures and net-hauls contained 18-79 pg YTXs per cell. Dilution series and analyses of cells from non-YTX-producing algal species demonstrated the presence of only minimal matrix effects on the ELISA, probably attributable to the presence of salts. The sensitivity of this method makes it possible to search for other possible producers of YTXs, and might also make it possible to follow the YTXs through the food chain. This method allows, for the first time, measurement of the variability in toxin content within a population of dinoflagellate cells--rather than just the average amount of toxin per cell.

Promoter variants in tissue inhibitor of metalloproteinase-3 (TIMP-3) protect against susceptibility in pigeon breeders' disease.

BACKGROUND: Tissue inhibitors of metalloproteinases (TIMPs) play a major role in extracellular matrix turnover in the lung. However, in chronic lung disorders such as idiopathic pulmonary fibrosis (IPF) and pigeon breeders' disease (PBD), TIMPs may promote an adverse non-degradative environment. We hypothesised that polymorphisms in TIMP-3 could affect susceptibility to IPF and PBD. METHODS: Two promoter variants, -915A>G and -1296T>C, were genotyped in 323 healthy subjects, 94 subjects with IPF, 115 with PBD, and 90 exposed to avian antigen but without PBD. The severity of fibrosis in lung tissue and the clinical outcome after 1 year was determined in the PBD group. RESULTS: The variants did not influence susceptibility to IPF, but the rare alleles of both variants appeared to be protective against susceptibility to PBD (odds ratio (OR) for carriage of at least one rare allele from either variant 0.48, 95% CI 0.30 to 0.76, p = 0.002). Haplotype analysis of positions -915 and -1296 estimated four haplotypes: *A*T, *G*T, *A*C and *G*C, respectively. Their frequencies differed overall between subjects with PBD and healthy subjects (p = 0.0049) and this was attributable primarily to the *G*C haplotype (OR 0.53, 95% CI 0.36 to 0.77, p = 0.001). The severity of fibrosis correlated with poorer outcome in the PBD group (r = 0.73, p<0.01) but no relationship was seen between the *G*C haplotype and outcome or fibrosis. However, PBD subjects with the *G*C haplotype did have proportionally fewer lymphocytes in their bronchoalveolar fluid than those with the common *A*T haplotype (p = 0.029). CONCLUSIONS: TIMP-3 variants appear to contribute to susceptibility to PBD. This may be through the inflammatory reaction rather than the fibrotic reaction.

Development of a highly sensitive screen for influenza A in guano and its application in the search for ancient RNA preserved under Antarctic Adelie penguin colonies.

We have developed a reverse transcriptase polymerase chain reaction (RT-PCR)-based assay to detect influenza A in guano samples as part of our program to investigate ancient viral RNA from under Antarctic Adelie penguin (Pygoscelis adeliae) colonies. Of five extraction protocols tested (RNeasy, GTC TRIZOL, GTC Silica, Rnaid, and AGPC), AGPC proved to be the most consistent and sensitive to low concentrations of influenza, but further purification with commercial viral nucleic acid spin filter system was still required to remove remaining PCR inhibitors. RT-PCR was then performed on the eluent and 650 bases of the M1 gene were amplified. The assay was found to be able to detect as little as 100 microl of 0.1 hemagglutination units (HU)/ml influenza.

Use of fluorescently labelled deoxyribonuclease I to spatially measure G-actin levels in migrating and non-migrating cells.

Lamellipodium protrusion is linked to actin filament disassembly in migrating fibroblasts [Cramer, 1999: Curr. Biol. 9:1095-1105]. To further study this relationship, we have identified a method to specifically and sensitively detect G-actin in distinct spatial locations in motile cells using deoxyribonuclease I (DNase I). Although DNase I can bind both G- and F-actin in vitro [Mannherz et al., 1980: Eur. J. Biochem. 95:377-385], when cells were fixed in formaldehyde and permeabilized in detergent, fluorescently-labelled DNase I specifically stained G-actin and not F-actin. 92-98% of actin molecules were stably retained in cells during fixation and permeabilization. Further, increasing or decreasing cellular G-actin concentration by treating live cells with latrunculin-A or jasplakinolide, respectively, caused a respective increase and decrease in DNase I cell-staining intensity as expected. These changes in DNase I fluorescence intensity accurately reflected increases and decreases in cellular G-actin concentration independently measured in lysates prepared from drug-treated live cells (regression coefficient = 0.98). This shows that DNase I cell-staining is very sensitive using this method. Applying this method, we found that the ratio of G-/F-actin is lower in both the lamellipodium and in a broad band immediately behind the lamellipodium in migrating compared to non-migrating fibroblasts. Thus, we predict that protrusion of the lamellipodium in migrating fibroblasts requires tight coupling to filament disassembly at least in part because G-actin is relatively limited within and behind the lamellipodium. This is the first report to directly demonstrate high sensitivity of cell-staining for any G-actin probe and this, together with the ready commercial accessibility of fluorescently-labelled DNase I, make it a simple, convenient, and sensitive tool for cell-staining of G-actin.

Male and female viewpoints on female circumcision in Ekpeye, Rivers State, Nigeria.

One hundred and ninety five male and female volunteers across the social strata were interviewed using structured questionnaire. Data were analysed using frequency tables. The study revealed that 74.7% of female respondents were circumcised. They believe that the practice would help prevent sexual promiscuity, curb sexual desires and that it is a custom they cannot do without. Most of the men would not marry an uncircumcised female, while a substantial number of the respondents would like to circumcise their daughters. Community effort to eradicate the practice is very minimal. Based on the findings, it is suggested that communities where female genital mutilation (FGM) is practiced as a social norm should be involved in eradication campaigns with support from national and international organisations.

Integrated enzyme-linked immunosorbent assay screening system for amnesic, neurotoxic, diarrhetic, and paralytic shellfish poisoning toxins found in New Zealand.

Enzyme-linked immunosorbent assays (ELISAs) were developed for amnesic, neurotoxic, and diarrhetic shellfish poisoning (ASP, NSP, and DSP) toxins and for yessotoxin. These assays, along with a commercially available paralytic shellfish poisoning (PSP) ELISA, were used to test the feasibility of an ELISA-based screening system. It was concluded that such a system to identify suspect shellfish samples, for subsequent analysis by methods approved by international regulatory authorities, is feasible. The assays had sufficient sensitivity and can be used on simple shellfish extracts. Alcohol extraction gave good recovery of all toxin groups. The ease of ELISAs permits the ready expansion of the system to screen for other toxins, as new ELISAs become available.

Novel properties of the protein kinase CK2-site-regulated nuclear- localization sequence of the interferon-induced nuclear factor IFI 16.

Members of the interferon-induced class of nuclear factors possess a putative CcN motif, comparable with that within proteins such as the simian virus 40 large tumour antigen (T-ag), which confers phosphorylation-mediated regulation of nuclear-localization sequence (NLS)-dependent nuclear import. Here we examine the functionality of the interferon-induced factor 16 (IFI 16) CcN motif, demonstrating its ability to target a heterologous protein to the nucleus, and to be phosphorylated specifically by the CcN-motif-phosphorylating protein kinase CK2 (CK2). The IFI 16 NLS, however, has novel properties, conferring ATP-dependent nuclear import completely independent of cytosolic factors, as well as binding to nuclear components. The IFI 16 NLS is not recognized with high affinity by the NLS-binding importin heterodimer, and transport mediated by it is insensitive to non-hydrolysable GTP analogues. The IFI 16 NLS thus mediates nuclear import through a pathway completely distinct from that of conventional NLSs, such as that of T-ag, but intriguingly resembling that of the NLS of the HIV-1 transactivator protein Tat. Since the IFI 16 CK2 site enhances nuclear import through facilitating binding to nuclear components, this represents a novel mechanism by which the site regulates nuclear-protein import, and constitutes a difference between the IFI 16 and Tat NLSs that may be of importance in the immune response.

Designing humanistic critical care environments.

The critical care environment can be designed to become more humanistic. Consideration of the environmental challenges of noise, lights, color, views, temperature, and comfort is essential. This article identifies the issues and concerns in the design of more humanistic healing in critical care units. Strategies to improve the environment include improving the physical and emotional tone of the unit through creative design, family and pet visitation, sleep promotion, and aromatherapy among others. In a life-threatening illness, attention paid to these concerns may significantly improve quality of life for patients and family.

Nuclear import of insulin-like growth factor-binding protein-3 and -5 is mediated by the importin beta subunit.

Although insulin-like growth factor-binding protein (IGFBP)-3 and IGFBP-5 are known to modulate cell growth by reversibly sequestering extracellular insulin-like growth factors, several reports have suggested that IGFBP-3, and possibly also IGFBP-5, have important insulin-like growth factor-independent effects on cell growth. These effects may be related to the putative nuclear actions of IGFBP-3 and IGFBP-5, which we have recently shown are transported to the nuclei of T47D breast cancer cells. We now describe the mechanism for nuclear import of IGFBP-3 and IGFBP-5. In digitonin-permeabilized cells, where the nuclear envelope remained intact, nuclear translocation of wild-type IGFBP-3 appears to occur by a nuclear localization sequence (NLS)-dependent pathway mediated principally by the importin beta nuclear transport factor and requiring both ATP and GTP hydrolysis. Under identical conditions, an NLS mutant form of IGFBP-3, IGFBP-3[(228)KGRKR --> MDGEA], was unable to translocate to the nucleus. In cells where both the plasma membrane and nuclear envelope were permeabilized, wild-type IGFBP-3, but not the mutant form, accumulated in the nucleus, implying that the NLS was also involved in mediating binding to nuclear components. By fusing wild-type and mutant forms of NLS sequences (IGFBP-3 [215-232] and IGFBP-5 [201-218]) to the green fluorescent protein, we identified the critical residues of the NLS necessary and sufficient for nuclear accumulation. Using a Western ligand binding assay, wild-type IGFBP-3 and IGFBP-5, but not an NLS mutant form of IGFBP-3, were shown to be recognized by importin beta and the alpha/beta heterodimer but only poorly by importin alpha. Together these results suggest that the NLSs within the C-terminal domain of IGFBP-3 and IGFBP-5 are required for importin-beta-dependent nuclear uptake and probably also accumulation through mediating binding to nuclear components.

Intranuclear binding by the HIV-1 regulatory protein VPR is dependent on cytosolic factors.

The regulatory protein Vpr of the human immunodeficiency virus HIV-1 performs multiple functions during the HIV replicative cycle. It is involved in the transport of the viral preintegration complex into the nucleus, and has the ability to interact with nuclear proteins such as transcription factors and cyclin-dependent kinases. In this study we examine for the first time the kinetics of intranuclear binding and accumulation at the nuclear envelope of fluorescently labelled full-length Vpr in vitro. We show that intranuclear binding is strongly dependent on the presence of cytosolic factors; in the absence of cytosol, Vpr associates predominantly with the nuclear envelope. Specific regulation of the interactions of Vpr with cytosolic factors, as well as with sites at the nuclear envelope and within the nucleus, is thus implicated, but conventional nuclear transport factors such as importin alpha/beta do not appear to be involved.