Long-Read Sequencing Improves Recovery of Picoeukaryotic Genomes and Zooplankton Marker Genes from Marine Metagenomes.

Long-read sequencing offers the potential to improve metagenome assemblies and provide more robust assessments of microbial community composition and function than short-read sequencing. We applied Pacific Biosciences (PacBio) CCS (circular consensus sequencing) HiFi shotgun sequencing to 14 marine water column samples and compared the results with those for short-read metagenomes from the corresponding environmental DNA samples. We found that long-read metagenomes varied widely in quality and biological information. The community compositions of the corresponding long- and short-read metagenomes were frequently dissimilar, suggesting higher stochasticity and/or bias associated with PacBio sequencing. Long reads provided few improvements to the assembly qualities, gene annotations, and prokaryotic metagenome-assembled genome (MAG) binning results. However, only long reads produced high-quality eukaryotic MAGs and contigs containing complete zooplankton marker gene sequences. These results suggest that high-quality long-read metagenomes can improve marine community composition analyses and provide important insight into eukaryotic phyto- and zooplankton genetics, but the benefits may be outweighed by the inconsistent data quality. IMPORTANCE Ocean microbes provide critical ecosystem services, but most remain uncultivated. Their communities can be studied through shotgun metagenomic sequencing and bioinformatic analyses, including binning draft microbial genomes. However, most sequencing to date has been done using short-read technology, which rarely yields genome sequences of key microbes like SAR11. Long-read sequencing can improve metagenome assemblies but is hampered by technological shortcomings and high costs. In this study, we compared long- and short-read sequencing of marine metagenomes. We found a wide range of long-read metagenome qualities and minimal improvements to microbiome analyses. However, long reads generated draft genomes of eukaryotic algal species and provided full-length marker gene sequences of zooplankton species, including krill and copepods. These results suggest that long-read sequencing can provide greater genetic insight into the wide diversity of eukaryotic phyto- and zooplankton that interact as part of and with the marine microbiome.

Tubo-ovarian abscess in virginal adolescent females: a case report and review of the literature.

BACKGROUND: A tubo-ovarian abscess (TOA) is a serious complication of pelvic inflammatory disease (PID), predominantly polymicrobial and present in sexually active women. TOA in virginal adolescent females are extremely rare but have serious and lifelong consequences. CASE: A 13 y.o. virginal female presented to the Emergency Room of a tertiary care pediatric hospital with abdominal pain and vomiting. Imaging suggested bowel compromise with potential perforation. An exploratory laparotomy revealed TOA which grew Escherichia Coli. This is the first reported case of Escherichia Coli TOA due to suspected bowel translocation. CONCLUSION: Review of the literature identified 8 cases of TOA in virginal adolescents. Given the severity of outcomes following TOA, this pathology should be considered in the differential diagnosis of virginal adolescents who present with fever and abdominal pain. If suspected, a prompt gynecology consult should be initiated, followed by a first line antibiotic therapy and when indicated, surgical drainage.

Microbial removal of atmospheric carbon tetrachloride in bulk aerobic soils.

Atmospheric concentrations of carbon tetrachloride (CCl(4)) were removed by bulk aerobic soils from tropical, subtropical, and boreal environments. Removal was observed in all tested soil types, indicating that the process was widespread. The flux measured in field chamber experiments was 0.24 ± 0.10 nmol CCl(4) (m(2) day)(-1) (average ± standard deviation [SD]; n = 282). Removal of CCl(4) and removal of methane (CH(4)) were compared to explore whether the two processes were linked. Removal of both gases was halted in laboratory samples that were autoclaved, dry heated, or incubated in the presence of mercuric chloride (HgCl(2)). In marl soils, treatment with antibiotics such as tetracycline and streptomycin caused partial inhibition of CCl(4) (50%) and CH(4) (76%) removal, but removal was not affected in soils treated with nystatin or myxothiazol. These data indicated that bacteria contributed to the soil removal of CCl(4) and that microeukaryotes may not have played a significant role. Amendments of methanol, acetate, and succinate to soil samples enhanced CCl(4) removal by 59%, 293%, and 72%, respectively. Additions of a variety of inhibitors and substrates indicated that nitrification, methanogenesis, or biological reduction of nitrate, nitrous oxide, or sulfate (e.g., occurring in possible anoxic microzones) did not play a significant role in the removal of CCl(4). Methyl fluoride inhibited removal of CH(4) but not CCl(4), indicating that CH(4) and CCl(4) removals were not directly linked. Furthermore, CCl(4) removal was not affected in soils amended with copper sulfate or methane, supporting the results with MeF and suggesting that the observed CCl(4) removal was not significantly mediated by methanotrophs.

Faecal indicator bacteria enumeration in beach sand: a comparison study of extraction methods in medium to coarse sands.

AIMS: The absence of standardized methods for quantifying faecal indicator bacteria (FIB) in sand hinders comparison of results across studies. The purpose of the study was to compare methods for extraction of faecal bacteria from sands and recommend a standardized extraction technique. METHODS AND RESULTS: Twenty-two methods of extracting enterococci and Escherichia coli from sand were evaluated, including multiple permutations of hand shaking, mechanical shaking, blending, sonication, number of rinses, settling time, eluant-to-sand ratio, eluant composition, prefiltration and type of decantation. Tests were performed on sands from California, Florida and Lake Michigan. Most extraction parameters did not significantly affect bacterial enumeration. anova revealed significant effects of eluant composition and blending; with both sodium metaphosphate buffer and blending producing reduced counts. CONCLUSIONS: The simplest extraction method that produced the highest FIB recoveries consisted of 2 min of hand shaking in phosphate-buffered saline or deionized water, a 30-s settling time, one-rinse step and a 10 : 1 eluant volume to sand weight ratio. This result was consistent across the sand compositions tested in this study but could vary for other sand types. SIGNIFICANCE AND IMPACT OF THE STUDY: Method standardization will improve the understanding of how sands affect surface water quality.

Performance of CHROMagar Staph aureus and CHROMagar MRSA for detection of Staphylococcus aureus in seawater and beach sand--comparison of culture, agglutination, and molecular analyses.

Beach seawater and sand were analyzed for Staphylococcus aureus and methicillin resistant S. aureus (MRSA) for samples collected from Avalon, and Doheny Beach, CA. Membrane filtration followed by incubation on CHROMagar Staph aureus (SCA) and CHROMagar MRSA (C-MRSA) was used to enumerate S. aureus and MRSA, respectively. Media performance was evaluated by comparing identification via colony morphology and latex agglutination tests to PCR (clfA, 16S, and mecA genes). Due to background color and crowding, picking colonies from membrane filters and streaking for isolation were sometimes necessary. The specificity of SCA and C-MRSA was improved if colony isolates were identified by the presence of a matte halo in addition to mauve color; however routine agglutination testing of isolates did not appear warranted. Using the appearance of a colony on the membrane filter in conjunction with isolate appearance, the positive % agreement, the negative % agreement, and the % positive predictive accuracy for SCA was 84%, 95%, and 99% respectively, and for C-MRSA it was 85%, 98%, and 92%, respectively. Sensitivity and specificity of SCA and C-MRSA with membrane-filtered beach samples were optimized through identification experience, control of filter volume and incubation time, and isolation of colonies needing further identification. With optimization, SCA and C-MRSA could be used for enumeration of S. aureus and MRSA from samples of beach water and sand. For the sites studied here, the frequency of detection of S. aureus ranged from 60 to 76% and 53 to 79% for samples of beach seawater and sand, respectively. The frequency of detection of MRSA ranged from 2 to 9% and 0 to 12% for samples of seawater and sand, respectively.

Pancreatic islet isolation variables in non-human primates (rhesus macaques).

BACKGROUND: Non-human primates (NHPs) are important preclinical models for pancreatic islet transplantation (PIT) because of their close phylogenetic and immunological relationship with humans. However, low availability of NHP tissue, long learning curves and prohibitive expenses constrain the consistency of isolated NHP islets for PIT studies. To advance preclinical studies, we attempted to identify key variables that consistently influence the quantity and quality of NHP islets. METHODS: Seventy-two consecutive pancreatic islet isolations from rhesus macaques were reviewed retrospectively. A scaled down, semi-automated islet isolation method was used, and monkeys with streptozotocin-induced diabetes, weighing 3-7 kg, served as recipients for allotransplantation. We analysed the effects of 22 independent variables grouped as donor factors, surgical factors and isolation technique factors. Islet yields, success of isolation and transplantation results were used as quantitative and qualitative outcomes. RESULTS: In the multivariate analysis, variables that significantly affected islet yield were the type of monkey, pancreas preservation, enzyme lot and volume of enzyme delivered. The variables associated with successful isolation were the enzyme lot and volume delivered. The transplant result was correlated with pancreas preservation, enzyme lot, endotoxin levels and COBE collection method. CONCLUSIONS: Islet quantity and quality are highly variable between isolations. The data reviewed suggest that future NHP isolations should use bilayer preservation, infuse more than 80 ml of Liberase into the pancreas, collect non-fractioned tissue from the COBE, and strictly monitor for infection.

A fold-back single-chain diabody format enhances the bioactivity of an anti-monkey CD3 recombinant diphtheria toxin-based immunotoxin.

T-cell depleting anti-CD3 immunotoxins have utility in non-human primate models of transplantation tolerance and autoimmune disease therapy. We recently reported that an affinity matured single-chain (scFv) anti-monkey CD3 antibody, C207, had increased binding to T-cells and increased bioactivity in a diphtheria toxin (DT)-based biscFv immunotoxin compared with the parental antibody, FN18. However, FN18 scFvs and their mutant derivatives such as C207 did not exhibit robust bivalent character in the biscFv format. We now report that C207 in a diabody format exhibits a 7-fold increase in binding to T-cells over scFv (C207) indicating considerable divalent character for the diabody. This construct was formed by reducing the V(L)/V(H) linker to five residues and was secreted from Pichia pastoris as the non-covalent dimer. An immunotoxin based on this diabody format was secreted as a non-covalent dimer but was devoid of bioactivity and failed to bind T-cells, suggesting steric hindrance from the two large closely positioned truncated DT moieties. We constructed a single-chain diabody immunotoxin by fusing to the truncated DT C-terminus L1-VL-L1-VH-L2-VL-L1-VH where L1 is a five-residue linker and L2 is the longer (G4S)3 linker permitting interactions between the distal and proximal VL/VH domains. This 'fold-back' immunotoxin was secreted predominantly as the monomer and exhibited a 5- to 7-fold increase in bioactivity over DT390biscFv(C207) and depleted monkey T-cells in vivo.

Polymorphisms of CD3epsilon in cynomolgus and rhesus monkeys and their relevance to anti-CD3 antibodies and immunotoxins.

The monoclonal antibody FN18 has been used as a marker for monkey T cells and as a T-cell-depleting reagent when conjugated to diphtheria toxin that was mutated to prevent binding to non-targeted cells. The antibody recognizes a conformational epitope on the ectodomain of monkey CD3epsilon and displays a range of binding activity to the T cells from different rhesus and cynomolgus monkeys. Our quantitative fluorescence-activated cell sorting analysis of the FN18 reactivity to T cells from different rhesus and cynomolgus monkeys showed that there are at least three levels of FN18 reactivity in the monkeys tested: high, moderate and low. On the basis of available DNA sequence information, we determined the gene structure of rhesus CD3epsilon chain and designed primers that can be used to amplify and quickly sequence the ectodomain of monkey CD3epsilon. Our sequence analysis revealed that the extent of nucleotide sequence variation in this area is greater than that previously reported. In addition to the amino acids at positions 45 and 50, we demonstrated that position 35 of CD3epsilon was also important and substitution of amino acid A for V at this position greatly reduced T-cell reactivity to FN18. We found that T cells from monkeys with high FN18 reactivity all had V, E and R at positions 35, 45 and 50 in CD3epsilon, respectively; those having low FN18 reactivity were homozygous in CD3epsilon with at least one of the changes: V35 to A, E45 to G and R to 50Q, whereas members in the moderate group are heterozygous, having both V and A, E and G, R and Q at these locations. A cytotoxicity assay revealed that T cells from a heterozygous rhesus monkey with moderate FN18 reactivity were much (about 40 times) less sensitive to a FN18-derived immunotoxin than those from a homozygous rhesus monkey having high FN18 reactivity.

Subcutaneous pancreatic islet transplantation using fibrin glue as a carrier.

BACKGROUND: Pancreatic islet grafts are difficult to manipulate and implant in the recipient site mainly because they are formed by a group of cells suspended in a solution. This physical property determines various characteristics that are unique for pancreatic islet transplantation. The purpose of this study was to evaluate the role of fibrin glue as a delivery method for islet transplantation. METHODS: C3H mouse islets were syngeneically transplanted into streptozotocin-diabetic recipients using fibrin glue in a subcutaneous pocket (Group 1) and using liquid islets injected under the kidney capsule (Group 2). Blood glucose levels were measured during 4 weeks of follow-up and compared against normal (Group 3) and diabetic levels (Group 4). RESULTS: No statistical differences were observed between the normal, kidney capsule, and fibrin glue groups. Only the diabetic group had a statistical difference when compared with the normal control group (P < .01). At the beginning, levels in Group 1 (fibrin glue) were higher than in Group 2 (kidney capsule), but turned into similar values after time and no statistical differences were observed between them during follow-up. CONCLUSIONS: Islet/fibrin glue grafts placed in a subcutaneous pocket obtained the same results as liquid grafts placed under the kidney capsule, proving to be an adequate delivery method for islet transplantation and solving some of the engraftment problems we find with liquid grafts.

RUNX1 associates with histone deacetylases and SUV39H1 to repress transcription.

RUNX1 (AML1) is a gene that is frequently disrupted by chromosomal translocations in acute leukemia. Like its Drosophila homolog Runt, RUNX1 both activates and represses transcription. Both Runt and RUNX1 are required for gene silencing during development and a central domain of RUNX1, termed repression domain 2 (RD2), was defined as being required for transcriptional repression and for the silencing of CD4 during T-cell maturation in thymic organ cultures. Although transcriptional co-repressors are known to contact other repression domains in RUNX1, the factors that bind to RD2 had not been defined. Therefore, we tested whether RD2 contacts histone-modifying enzymes that may mediate both repression and gene silencing. We found that RD2 contacts SUV39H1, a histone methyltransferase, via two motifs and that endogenous Suv39h1 associates with a Runx1-regulated repression element in murine erythroleukemia cells. In addition, one of these SUV39H1-binding motifs is also sufficient for binding to histone deacetylases 1 and 3, and both of these domains are required for full RUNX1-mediated transcriptional repression. The association between RUNX1, histone deacetylases and SUV39H1 provides a molecular mechanism for repression and possibly gene silencing mediated by RUNX1.

How has the 'two-week wait' rule affected the presentation of colorectal cancer?

OBJECTIVE: To assess the impact of the 'two-week wait' rule on the presentation of colorectal cancer. METHODS: A retrospective study of all patients referred to a fast-track clinic in a colorectal cancer centre over an 18-month period, documenting outcome, especially colorectal cancer diagnosis. Comparison was made with patients diagnosed with colorectal cancer presenting via other routes in the same time period. RESULTS: Over an 18-month period, 462 patients were seen in the fast-track clinic and 64 (13.8%) were diagnosed with colorectal cancer. A further 131 patients with colorectal cancer presented to the department in the same time period through other means; 66 via standard out-patient letters, 26 from other departments and 39 (20%) as emergency admissions. Median (range) time to first clinic was 12 (2-28) days for fast-track and 24 (1-118) days for standard referrals (P < 0.0001); median time to first treatment was a further 36 (9-134) and 36.5 (1-226) days, respectively. The fast-track cohort had more advanced staging than those referred by standard letter. There were 19 Dukes' B, 22 Dukes' C and 14 Dukes' D cancers in the fast-track group compared with 28 Dukes' B, 25 Dukes' C and 6 Dukes' D in the standard referral group. After patient interview, only 337 (73%) of 462 fast-track patients appeared to fulfil the referral criteria but of the 64 diagnosed with cancer, 59 (92%) satisfied the criteria. Of the 66 patients with cancer referred by standard letter, 61 (92%) fulfilled the criteria. CONCLUSION: Patients referred to the fast-track clinic were seen quicker than those referred by standard letter, but they tended to have more advanced disease. The fast-track referral criteria were fulfilled by most patients with cancer (whether or not they were referred to the fast track clinic), confirming their validity. After detailed interview in the clinic, a quarter of fast-track referrals were found not to satisfy referral criteria, suggesting that prioritization in primary care could be improved.

Pneumonic lesions in lambs in New Zealand: patterns of prevalence and effects on production.

AIM: To describe temporal and spatial patterns of the prevalence of pneumonia in lambs in three regions of New Zealand, and determine effects on liveweight gain. METHODS: A longitudinal study was conducted in 1999- 2000 on 14 commercial sheep farms, five from the Southland region of the South Island, and five from the King Country and four from the Northland regions of the North Island. Pneumonia was known to be a problem in lambs on all farms selected. On each farm, 400 lambs were randomly selected at weaning and allocated at random to one of two groups (Groups I and II). Lambs in both groups were weighed at 4-weekly intervals. The extent of pneumonic lesions in lungs was scored visually at slaughter in 40 randomly selected lambs after each weighing (Group I). Lambs in Group II were selected for slaughter by the farm manager on a commercial basis of liveweight and body condition, then assessed for pneumonic lesions. RESULTS: Prevalence of extensive pneumonic lesions (>/=10% lung surface area affected) increased from December to March from 1.4 to 10.1% lambs in Southland (n=1,917), 1.2 to 25.7% in the King Country (n=1,831), and 4.7 to 21.4% in Northland (n=1,485). Overall, 42.3% of lambs in both groups had pneumonic lesions (n=5,233) and in 15.1% of these, >/=5% of the lung surface area was affected. Daily weight gain in the month prior to slaughter was reduced in lambs for which >/=20% lung surface area was affected in Group I (p<0.05) but not in Group II. CONCLUSIONS: The association between pneumonic lesions and reduced weight gain was highly significant in lambs from Group I, but not from Group II. Thus, pneumonia had a negative impact on weight gain, and selection by farmers of lambs for slaughter counteracted this association in lambs from Group II. CLINICAL RELEVANCE: Pneumonia occurs commonly as a subclinical disease in lambs in New Zealand. It has no public health significance and is not recorded at slaughter plants nor reported to farmer clients. Effects of the disease on daily weight gain of lambs is considerable and costly for lamb producers.

New Zealand veterinarians--demography, remuneration and vacancies.

AIMS: To determine levels of remuneration for veterinarians in New Zealand, to examine associations between putative explanatory factors and gross annual remuneration, and to quantify the type and prevalence of vacant positions. METHODS: A postal survey to 486 identifiable clinical practices and 53 identifiable organisations that employ veterinarians was used to gather data for the 2-month period of December 2001 to January 2002. RESULTS: Data were produced for 972 veterinarians (367 females and 605 males) working in 325 clinical practices, and 299 veterinarians (88 females and 211 males) employed by 32 organisations. Median levels of gross annual remuneration for assistants, partners/ shareholders and sole owners working >/=5 days per week in clinical practice were NZ$60,000, $90,000 and $75,000, respectively, and for veterinarians in organisations, irrespective of number of days per week worked, was $68,000. Pay rates increased linearly as the number of years since graduation increased for all clinicians and with increasing age for veterinarians in organisations. Full-time assistants were likely to be paid more if the practice was rural rather than urban in location, if they were males, and if administrative duties were part of the job. The same factors, except for sex, were significant for remuneration for owners and partners/shareholders working full-time. Their remuneration tended to be higher if the practice was involved with either dairy or deer work but decreased as the number of animal species serviced increased and if they worked >5 days per week. Part-time female veterinarians were generally paid more than male counterparts. Male veterinarians working in organisations were generally paid about 8% more than their female colleagues. Veterinarians in organisations involved with administration at a head office were generally better paid than those without administrative duties. Pay rates were, on the whole, better in private organisations than in universities, state-owned enterprises, government-operated and other types of organisations About 50% of all services provided by clinical practices were directed to small animals, 27% to dairy cattle and about 10%, 6% and 3% to horses, sheep and beef cattle, and deer, respectively. About 31% of veterinarians worked solely with small animals but most had multiple species workloads. Of the 325 respondent practices, 98 reported vacancies for 119 veterinarians, of which 79 were full-time, 27 part-time and 12 locum positions. Of the 32 respondent organisations, seven reported vacancies for 16 mostly full-time positions. Farmer owned co-operative practices were less likely than privately owned practices to have full-time vacant positions. The only factor identified as influencing part-time vacancies in clinical practices was hourly pay rate. Vacancies occurred randomly across practices, irrespective of location, and there was no indication of greater demand for services for any particular species. The odds of a vacancy in organisations was lower for state-owned enterprises and private organisations than for government organisations (odds ratios (OR)=0.14 and 0.18, respectively). CONCLUSIONS: Relatively more females than males worked part-time and 23% of all assistants in clinical practice worked part-time. Sex made a significant difference to gross remuneration for full-time assistants in clinical practice and for veterinarians employed by private or government organisations. In both situations, males were generally better paid than females. Female part-time assistants and partners/shareholders or sole owners in clinical practice were generally better rewarded than their male counterparts. Sex had no effect on remuneration levels for owners/ partners working full-time in clinical practices. The study confirmed a serious shortage of veterinarians in New Zealand. The probability of a vacancy occurring in farmer owned co-operative ('club') practices was lower than in private practices. Vacancies were distributed randomly among rural, urban and rural/urban practices with no evidence of rural practices being more severely affected than urban or rural/urban practices.

Optimal thresholds of the lecithin/sphingomyelin ratio and lamellar body count for the prediction of the presence of phosphatidyl glycerol in diabetic women.

OBJECTIVE: To assess the optimal thresholds of the lecithin/sphingomyelin (L/S) ratio and lamellar body count for the prediction of the presence of phosphatidyl glycerol (PG) in diabetic pregnant women. METHODS: We accessed a database of clear amniotic fluid specimens obtained by transabdominal amniocentesis in diabetic women with singleton non-malformed fetuses. PG results were classified as 'absent' or 'present'. Receiver operating characteristic (ROC) curve analysis was constructed of different L/S ratios and lamellar body counts to identify the optimal threshold for prediction of the presence of PG. Sensitivity was defined as the rate of L/S ratio and lamellar body count above specific thresholds among cases with present PG. The false-positive rate was that of L/S ratios or lamellar body counts above specific thresholds among cases with absent PG. Statistical analysis included one-way analysis of variance with post-hoc analysis, with p < 0.05 considered significant. RESULTS: A total of 76 consecutive women were included in the analysis, 74% (n = 56) using insulin and the remainder treated by diet alone. L/S and PG results were both available in 72 women. PG was reported as 'present' in 70% (51/73) of specimens. As expected, there was a significant relationship between L/S ratios and presence of PG (area under the curve = 0.932, p < 0.001). An L/S ratio of > or = 3.0 represented the optimal trade-off between sensitivity (68%) and false-positive rate (6%) in the prediction of present PG. Similarly, there was a significant relationship between lamellar body count values and presence of PG (area under the curve = 0.976, p < 0.001). A lamellar body count of > or = 50 000 represented the optimal trade-off between sensitivity (92%) and false-positive rate (0%) in the prediction of present PG. CONCLUSION: In diabetic pregnant patients, the presence of PG in the amniotic fluid more closely corresponded to an L/S ratio of > or = 3.0 or to a lamellar body count of > or = 50,000.

Toward a model of false recall: experimental manipulation of encoding context and the collection of verbal reports.

The likelihood of false recall in the Deese-Roediger-McDermott (DRM) paradigm was shown to depend on encoding context in two experiments. When fillers had been preselected to decrease the likelihood of encoding the critical lure's semantic features, false recall was virtually eliminated. However, when the same words were presented rearranged in different presentation orders, levels of false recall that were found in earlier DRM studies (Robinson & Roediger, 1997) were replicated. The role of encoding processes in the DRM paradigm was further explored with additional participants completing the experiment while thinking aloud. During encoding of word lists, participants verbalized semantic elaboration of the critical lure while studying the word lists. A path analysis demonstrated that participants' verbalization of critical lures during encoding reliably predicted their level of false recall.

Consumption of tropospheric levels of methyl bromide by C(1) compound-utilizing bacteria and comparison to saturation kinetics.

Pure cultures of methylotrophs and methanotrophs are known to oxidize methyl bromide (MeBr); however, their ability to oxidize tropospheric concentrations (parts per trillion by volume [pptv]) has not been tested. Methylotrophs and methanotrophs were able to consume MeBr provided at levels that mimicked the tropospheric mixing ratio of MeBr (12 pptv) at equilibrium with surface waters ( approximately 2 pM). Kinetic investigations using picomolar concentrations of MeBr in a continuously stirred tank reactor (CSTR) were performed using strain IMB-1 and Leisingeria methylohalidivorans strain MB2(T) - terrestrial and marine methylotrophs capable of halorespiration. First-order uptake of MeBr with no indication of threshold was observed for both strains. Strain MB2(T) displayed saturation kinetics in batch experiments using micromolar MeBr concentrations, with an apparent K(s) of 2.4 microM MeBr and a V(max) of 1.6 nmol h(-1) (10(6) cells)(-1). Apparent first-order degradation rate constants measured with the CSTR were consistent with kinetic parameters determined in batch experiments, which used 35- to 1 x 10(7)-fold-higher MeBr concentrations. Ruegeria algicola (a phylogenetic relative of strain MB2(T)), the common heterotrophs Escherichia coli and Bacillus pumilus, and a toluene oxidizer, Pseudomonas mendocina KR1, were also tested. These bacteria showed no significant consumption of 12 pptv MeBr; thus, the ability to consume ambient mixing ratios of MeBr was limited to C(1) compound-oxidizing bacteria in this study. Aerobic C(1) bacteria may provide model organisms for the biological oxidation of tropospheric MeBr in soils and waters.

High maternal serum inhibin A levels following the loss of one fetus in a twin pregnancy.

Inhibin A levels are elevated in the second trimester of pregnancies affected with fetal Down syndrome, on average, two times the level in unaffected pregnancies. Inhibin A levels are also two times higher in twin than in singleton pregnancies. Prenatal serum screening using inhibin A levels as a second trimester marker began at the Women and Infants Hospital in March 1998. We describe a case of a 17-year-old woman thought to have had a complete spontaneous abortion of a twin pregnancy but later found to be continuing the pregnancy with a single fetus. Routine second trimester prenatal serum screening revealed an extremely elevated inhibin A level of 39 MoM (multiples of the median). The patient delivered an apparently healthy female infant at 41 weeks of gestation. Therefore, inhibin A may be extremely elevated in the second trimester of a twin pregnancy after the loss of one fetus and this increased inhibin A level does not have any obvious adverse maternal or fetal effects.

Use of capillary electrophoresis and fluorescent labeled peptides to detect the abnormal prion protein in the blood of animals that are infected with a transmissible spongiform encephalopathy.

Transmissible spongiform encephalopathies in humans and in animals are fatal neuro-degenerative diseases with long incubation times. The putative cause of these diseases is a normal host protein, the prion protein, that becomes altered. This abnormal prion protein is found mostly in the brains of infected individuals in later stages of the disease, but also can be found in lymphoid and other tissues in lower amounts. In order to eradicate this disease in animals, it is important to develop a system that can concentrate the abnormal prion protein and an assay that is very sensitive. The sensitivity that can be achieved with capillary electrophoresis makes it possible to detect the abnormal protein in blood. A peptide from the carboxyl terminal region, amino acid positions 218-232, was labeled with fluorescein during the synthesis of the peptide at the amino terminus. Antibodies that have been produced to this peptide were affinity purified and used in a capillary electrophoresis immunoassay. The amount of fluorescein labeled peptide in the capillary was 50 amol. Blood was obtained from normal sheep and elk, from sheep infected with scrapie and elk infected with chronic wasting disease. Buffy coats and plasma were prepared by a conventional method. After treatment with proteinase K, which destroys the normal protein but not the altered one, the blood fractions were extracted and tested in the capillary electrophoresis immunoassay for the abnormal prion protein. The abnormal prion protein was detected in fractions from blood from infected animals but not from normal animals. This assay makes a pre-clinical assay possible for these diseases and could be adapted to test for the abnormal prion protein in process materials that are used for manufacture of pharmaceuticals and products for human consumption.

Is bipolar disorder still underdiagnosed? Are antidepressants overutilized?

BACKGROUND: Previous studies have suggested that bipolar disorder may be underdiagnosed, and that antidepressants may be over-utilized in its treatment. METHODS: Consecutively admitted patients (n =48) diagnosed with DSM-IV bipolar disorder, type I, (n = 44) or schizoaffective disorder, bipolar type, (n = 4) were interviewed systematically and their charts were reviewed to confirm diagnosis before admission. They were then treated according to systematic structured interview diagnoses. These data reflect the changes in diagnoses and treatment. RESULTS: 40% (19/48) were identified with previously undiagnosed bipolar disorder, all previously diagnosed with unipolar major depressive disorder. A period of 7.5+/-9.8 years elapsed in this group before bipolar diagnosis was made. Antidepressant use was high on admission (38%) and was reduced with acceptable treatment response rates. The adjunctive use of risperidone appeared to be a good treatment alternative. LIMITATIONS: While diagnoses were made prospectively, treatment response was assessed retrospectively, and was based on non-randomized, naturalistic therapy. CONCLUSIONS: Systematic application of DSM-IV criteria identified previously undiagnosed bipolar disorder in 40% of a referred population of patients with mood disorders, all previously misdiagnosed as unipolar major depressive disorder. Antidepressants appeared overutilized and risperidone was an effective alternative adjunctive therapy agent.