Global phylogenomic assessment of Leptoseris and Agaricia reveals substantial undescribed diversity at mesophotic depths.

BACKGROUND: Mesophotic coral communities are increasingly gaining attention for the unique biological diversity they host, exemplified by the numerous mesophotic fish species that continue to be discovered. In contrast, many of the photosynthetic scleractinian corals observed at mesophotic depths are assumed to be depth-generalists, with very few species characterised as mesophotic-specialists. This presumed lack of a specialised community remains largely untested, as phylogenetic studies on corals have rarely included mesophotic samples and have long suffered from resolution issues associated with traditional sequence markers. RESULTS: Here, we used reduced-representation genome sequencing to conduct a phylogenomic assessment of the two dominant mesophotic genera of plating corals in the Indo-Pacific and Western Atlantic, respectively, Leptoseris and Agaricia. While these genome-wide phylogenies broadly corroborated the morphological taxonomy, they also exposed deep divergences within the two genera and undescribed diversity across the current taxonomic species. Five of the eight focal species consisted of at least two sympatric and genetically distinct lineages, which were consistently detected across different methods. CONCLUSIONS: The repeated observation of genetically divergent lineages associated with mesophotic depths highlights that there may be many more mesophotic-specialist coral species than currently acknowledged and that an urgent assessment of this largely unstudied biological diversity is warranted.

Applications for the new electrochemiluminescent (ECL) and biosensor technologies.

Biosensor and electrochemiluminescent (ECL) assays are replacing enzyme-linked immunosorbent assays (ELISAs) at Schering-Plough as immunoassays of choice to monitor cytokine levels and detect anti-cytokine antibody responses during cytokine therapy. These new assays provide increased sensitivity and a better correlation with biological assays. Biosensor assays using the BIACORE 2000 (BIACORE, Uppsala, Sweden) are being adopted to support preclinical and clinical trials for the detection of antibodies capable of binding to IL-10 and IL-4. Significant advantages when using a biosensor assay are that real-time and label-free detection permit increased throughput and direct detection of binding interactions which enables detection of low affinity antibodies that are not detected by ELISA. The ECL assays using the ORIGEN Analyser (IGEN, Gaithersburg, MD) that we have implemented to replace existing ELISAs for quantification of serum IL-10 and serum interferon alfa levels are more sensitive and less subject to matrix effects. Data obtained during the validation of these assays are described.

Detection and characterization of antibodies to PEG-IFN-alpha2b using surface plasmon resonance.

Some patients treated with type I interferon (IFN) preparations develop neutralizing antibodies that may abrogate any clinical benefit. We have a new complex of polyethylene glycol12000 and IFN-alpha2b (PEG-IFN-alpha2b) in clinical trials and need to be able to detect any antibodies formed specifically against the complex. We have, therefore, devised a method based on measurement of surface plasmon resonance (SPR) in the BIACORE 2000 apparatus. PEG-IFN-alpha2b is anchored to one flow cell on the sensor chip, IFN-alpha2b to another, and PEG to a third. A 20 microl serum sample flows in turn through the three cells, which are optically scanned. Any antibodies in the serum bind to the corresponding immobilized antigen, and a change in the optical signal is generated. With appropriate specific reagents, their immunoglobulin isotype can be similarly established. The automated assay can quickly test numerous sera. Very little serum is needed, and the assay is reliable and precise and can detect low-alphaffinity antibodies.

A highly sensitive electrochemiluminescence immunoassay for interferon alfa-2b in human serum.

A highly sensitive immunoassay for the quantitative measurement of recombinant interferon alfa-2b (IFN alfa-2b) in serum was developed using the ORIGEN electrochemiluminescence (ECL) detection system. The ECL assay developed uses a ruthenylated mouse monoclonal anti-IFN-alfa antibody and a sheep polyclonal anti-IFN-alfa antibody that has been biotinylated. An immune complex, formed between the antibodies and the IFN in the serum, is captured by streptavidin coated paramagnetic beads. The assay is sensitive and can accurately measure 4 IU/ml in undiluted serum samples. In addition, this assay requires less labor than classical ELISAs and is not significantly affected by individual serum factors. The total assay variance for both intra- and inter-assay precision is < 10%. The assay is specific for the analyte. Mean percent recoveries in pooled and individual donor serum samples range from 84 to 114%. In addition, results of the ECL assay correlate well with a bioassay and were more accurate that an ELISA for the detection of interferon alfa-2b in individual human serum samples. The assay can be modified to measure other forms of the analyte and/or interferon in other matrices.

Impaired proliferation and tumorigenicity induced by CCAAT/enhancer-binding protein.

A plasmid containing the CCAAT/enhancer-binding protein (C/EBP alpha) gene transcriptionally controlled by the metallothionein promoter was constructed. The gene was transfected into the human hepatocellular carcinoma cell lines Hep3B and HepG2. When cultured in vitro in the presence of 100 microM ZnSO(4), C/EBP alpha expression caused reversible growth arrest. In soft agar clonogenic assays, C/EBP alpha expression decreased both the colony size and the total number of colonies compared with zinc-free controls. C/EBP alpha expressing cells s.c. implanted in CD-1 nu/nu mice were essentially nontumorigenic, whereas C/EBP alpha tumor cells implanted into immunodeficient SCID mice demonstrated a significantly delayed time of tumor appearance compared with cells transfected with a vector control plasmid. These studies suggest that the expression of endogenous genes normally associated with a quiescent, differentiated state, such as C/EBP alpha, can result in impaired proliferative activity and suppressed tumorigenicity of hepatoma cell lines.

The nature of the copper ions in the membranes containing the particulate methane monooxygenase from Methylococcus capsulatus (Bath).

It is shown that the particulate methane monooxygenase (pMMO) has an obligate requirement for copper. The MMO activity in the particulate fractions obtained from Methylococcus capsulatus (Bath) cells is found to increase with increasing copper content of the membranes. The enzyme activity from membranes obtained from cells grown at low copper levels can be stimulated further by the addition of Cu(II) ions to the assay medium. The membrane-bound copper ions can exist in both Cu(II) and Cu(I) forms. EPR and magnetic susceptibility characterizations of the membranes indicate the presence of an exchange-coupled trinuclear Cu(II) cluster when the bulk of the copper ions is oxidized. However, the functional form of the enzyme is the reduced or partially reduced form. The copper ions in the membrane fractions as isolated often exhibit a high level of reduction. An EPR spectrum with one unpaired electron spin delocalized over three copper nuclei has been observed for the two-electron reduced trinuclear copper cluster. The high correlation between the copper level in the membranes and enzymatic activity as well as the high reactivity of the reduced copper clusters toward dioxygen strongly indicate that the membrane-bound copper ions constitute the active sites of the pMMO.

Minimal antigenicity of intron A in human recipients demonstrated by three analytical methods.

Antibodies to recombinant human interferon alpha 2b (Intron A) were detected in only a small number of 101 Intron A recipients. This group of cancer patients received Intron A for a mean treatment time of 4.3 months and were selected from disease categories in which subjects were expected to be immunocompetent. Three methods for the detection of antibodies were employed: (a) a bioassay measuring the neutralizing activity of the sera for the antiviral action of interferon alpha 2b, (b) a radioimmunological assay measuring the ability of the sera to prevent the detection of interferon alpha 2b by an immunoradiometric assay (IRMA), and (c) an enzyme-linked immunosorbent assay (ELISA) that measures the binding of immunoglobulins to interferon alpha 2b attached to a solid support. Three of the 101 patients developed neutralizing activity during treatment. Two of these exhibited low neutralizing titers of 1:6-1:9 and were unreactive in the IRMA and ELISA, while only one was positive by bioassay, IRMA, and ELISA. An additional seven patients were positive only in the ELISA. Six of these were borderline positive, i.e., the posttreatment:pretreatment ratio was less than or equal to 5. The results of this study confirm that Intron A is minimally antigenic in human subjects.

Polarographic cerebral oxygen availability, fluorocarbon blood levels and efficacy of oxygen transport by emulsions.

In order to relate blood perfluorocarbon (PFC) level to brain tissue oxygen availability (aO2) and respiratory oxygen (FIO2), twelve conscious rabbits with chronically implanted platinum cathodes were infused with six types of emulsions in 14 infusions and their response to oxygen and carbogen breathing recorded. Blood was removed for sampling and to avoid a volume overload. Blood lactate was measured as an indicator of adequacy of perfusion. Glucose, osmotic pressure, packed cell volume, PFC by combustion and volatilization were also measured in blood samples. Methyl prednisolone was administered to 5 rabbits. Blood PFC levels measured by the commonly used centrifugation method (Fluorocrit) were subject to considerable variation, depending mainly upon centrifugation time. Fluorocrit values after Oxypherol infusion decreased from an average of 43% for 5 minutes to 15% for 30 minutes centrifugation. Fluorocrit tended to slightly increase with time of centrifugation when phospholipid was used as the emulsifier, early in PFC infusion and on the next day. Blood fluorocarbon was always lower, about half that of 30 minutes of centrifugation, when determined by combustion or by volatilization, than by centrifugation. The increase in brain a O# response to oxygen and carbogen was observed at a lower PFC blood level than expected and in some animals appeared in either the right or left hemisphere, but not in both, suggesting that oxygen transport, at least near the electrode, was by other than oxygen solubility in blood PFC. Blood lactate proved to be an excellent monitor of whole body perfusion. Animals with a blood lactate above 5 mM/L at 1 hour post infusion died the following day. The fluorocrit after 5, 10, 20, and 30 minutes of centrifugation, which we have named the "fluoro-gram," can have a sharp downward, a slight upward curve, or stay level, depending upon the type of emulsifier, the amount of PFC circulating and the time it has circulated. The fluorocarbon-induced increases in aO2 persist through the third day. This enhanced cerebrocortical aO2 current can be very roughly calculated as: aO2 current equals the air aO2 current plus (the 30 minute fluorocrit times K) where K for oxygen is 0.05 and for carbogen is 0.07. Some enhancement of the aO2 current in oxygen lingers to the fifth day after the blood PFC level is vanishingly small. Methyl prednisolone has a transient effect in suppressing the PFC enhanced oxygen and carbogen aO2 responses and no effect on the Oxypherol fluorogram.(ABSTRACT TRUNCATED AT 400 WORDS)

Injuries to runners: a study of entrants to a 10,000 meter race.

As the number of runners has increased dramatically, so has the incidence of running-related injuries. In order to determine what training factors are associated with running-related injuries, as well as what percentage of injured runners seeks professional medical attention, a random sample of entrants to a 10 kilometer race was asked to complete a questionnaire. There were 451 respondents, 355 men and 96 women, with a nonresponse rate of 12.7%. Nonrespondents did not differ from respondents with regard to age or sex. Forty-seven percent of respondents indicated that they had sustained a running-related injury in the last 2 years. Injured runners differed significantly from noninjured runners in that they were more likely to have run more miles per week, run more days per week, run a faster pace, run more races in the last year, stretched before running, and not participated regularly in other sports. Associated with injury, but not statistically significant, were those who had run marathons and had done muscle-strengthening exercises. No association was found with regard to the length of time running, running surfaces, part of the foot first contacting the ground, or running intervals, sprints, or hills. Seventy percent of those injured sought professional medical care, with 76% of these having a good or excellent recovery from their injuries. Compliance with medical advice correlated well with treatment success.