Artemis splice defects cause atypical SCID and can be restored in vitro by an antisense oligonucleotide.

Artemis deficiency is known to result in classical T-B- severe combined immunodeficiency (SCID) in case of Artemis null mutations, or Omenn's syndrome in case of hypomorphic mutations in the Artemis gene. We describe two unrelated patients with a relatively mild clinical T-B- SCID phenotype, caused by different homozygous Artemis splice-site mutations. The splice-site mutations concern either dysfunction of a 5' splice-site or an intronic point mutation creating a novel 3' splice-site, resulting in mutated Artemis protein with residual activity or low levels of wild type (WT) Artemis transcripts. During the first 10 years of life, the patients suffered from recurrent infections necessitating antibiotic prophylaxis and intravenous immunoglobulins. Both mutations resulted in increased ionizing radiation sensitivity and insufficient variable, diversity and joining (V(D)J) recombination, causing B-lymphopenia and exhaustion of the naive T-cell compartment. The patient with the novel 3' splice-site had progressive granulomatous skin lesions, which disappeared after stem cell transplantation (SCT). We showed that an alternative approach to SCT can, in principle, be used in this case; an antisense oligonucleotide (AON) covering the intronic mutation restored WT Artemis transcript levels and non-homologous end-joining pathway activity in the patient fibroblasts.

Experiences and potential of anaerobic wastewater treatment in tropical regions.

In this article an overview is given on the experience with anaerobic treatment of domestic wastewater in the tropics, with emphasis on the situation in India. Some design criteria and their impact on the costs of UASB reactors are discussed. The operational results of a number of full-scale reactors are presented. The applicability of the UASB, in combination with different post-treatment units to comply with a variety of effluent standards is compared with other systems. From the available data it is concluded that, if nitrogen removal is not required, a UASB system, followed by a polishing pond, is a relatively simple, affordable, and manageable wastewater treatment system. Trickling filters may follow UASB treatment if effluent standards require removal of TKN. The inclusion of UASB reactors in wastewater treatment schemes allowing for total nitrogen removal still needs further study.

Growth Characteristics of the Thermophilic Fungus Scytalidium thermophilum in Relation to Production of Mushroom Compost.

Scytalidium thermophilum is an important thermophilic fungus in the production of mushroom compost. I investigated the characteristics of this organism and present a simple model with which fungal growth in compost can be described. The model is used to predict better circumstances for rapid indoor production of mushroom compost. I conclude that inoculation of the starting material with prepared compost either before or after the pasteurization phase has only a minor effect on the shortening of the composting process. This is because the initial growth rate of the fungus is much higher than its growth rate later. A lower temperature (53.5 degrees C instead of the usual 56 to 58 degrees C) during the pasteurization phase may be most profitable for rapid compost production; such a temperature may reduce the time that is needed for the last phase of the production process by at least 1 day.

Thermophilic anaerobic digestion of high strength wastewaters.

Investigations on the thermophilic anaerobic treatment of high-strength wastewaters (14-65 kg COD/m(3)) are presented. Vinasse, the wastewater of alcohol distilleries, was used as an example of such wastewaters. Semicontinuously fed digestion experiments at high retention times revealed that the effluent quality of digestion at 55 degrees C is comparable with that at 30 degrees C at similar loading rates. The amount of methane formed per kilogram of vinasse drops almost linearly with increasing vinasse concentrations. This can be attributed to increasing concentrations of inhibitory compounds, resulting in increasing volatile fatty acid (VFA) concentrations in the effluent. The treatment of vinasse was also investigated using upflow anaerobic sludge blanket (UASB) reactors. Thermophilic granular sludge, cultivated on sucrose, was used as seed material. The sludge required a 4-month adaptation period, during which the size of the sludge granules decreased significantly. However, the settling characteristics remained satisfactory. After adaptation, high loading and methane generation rates could be accommodated at satisfactory treatment efficiencies, namely, 86.4 kg COD/m(3) day and 26 m(3) CH(4)(STP)/m(3) day, respectively. As in the semicontinuously fed digesters, the effluent VFA concentrations were virtually independent of the loading rates applied, indicating that the toxicity of the vinasse is more important than the loading rate in determining the efficiency of the conversion of vinasse to methane.

Granulation of biomass in thermophilic upflow anaerobic sludge blanket reactors treating acidified wastewaters.

The development of granular sludge in thermophilic (55 degrees C) upflow anaerobic sludge blanket reactors was investigated. Acetate and a mixture of acetate and butyrate were used as substrates, serving as models for acidified waste-waters. Granular sludge with either Methanothrix or Methanosarcina as the predominant acetate utilizing methanogen was cultivated by allowing the loading rate to increase whenever the acetate concentration in the effluent dropped below 200 and 700 mg COD/L, respectively. The highest methane generation rates, up to 162 kg CH(4)-COD/m(3) day, or 2.53 mole CH(4)/L day, were achieved at hydraulic retention times down to 21 min, with granules consisting of Methanothrix. The formation of Methanothrix granules did not depend on the type of seed material, nor on the addition of inert support particles. The growth of granules proceeded rapidly with adapted seed material, even when the reactors were inoculated with low concentrations. With mesophilic seed materials growth of granules took much longer. Thermophilic Methanothrix granules strongly resemble mesophilic granules of the "filamentous" type. Some factors governing the thermophilic granulation process are discussed.

Fermentation of Inulin by Clostridium thermosuccinogenes sp. nov., a Thermophilic Anaerobic Bacterium Isolated from Various Habitats.

Four closely related strains of thermophilic bacteria were isolated via enrichment in batch and continuous culture with inulin as the sole source of carbon and energy by using inoculations from various sources. These new strains were isolated from beet pulp from a sugar refinery, soil around a Jerusalem artichoke, fresh cow manure, and mud from a tropical pond in a botanical garden. The cells of this novel species of strictly anaerobic, gram-positive bacteria were rod shaped and nonmotile. Growth on inulin was possible between 40 and 65 degrees C, with optimum growth at 58 degrees C. All strains were capable of fermenting a large number of sugars. Formate, acetate, ethanol, lactate, H(2), and succinate were the main organic fermentation products after growth on fructose, glucose, or inulin. Synthesis of inulinase in batch culture closely paralleled growth, and the enzyme was almost completely cell bound. Strain IC is described as the type strain of a new species, Clostridium thermosuccinogenes sp. nov., with a G+C content of 35.9 mol%.

Growth-promoting effect of thermophilic fungi on the mycelium of the edible mushroom Agaricus bisporus.

The growth-promoting effect of the thermophilic fungus Scytalidium thermophilum in mushroom compost on the mycelium of the edible mushroom Agaricus bisporus was investigated. Results obtained by others were confirmed by showing that S. thermophilum leads to an increased hyphal extension rate of the mushroom mycelium. However, it was demonstrated that hyphal extension rates were not clearly related to mushroom biomass increase rates. A number of experiments pointed strongly towards CO2 as the determinant of hyphal extension rates. In compost, CO2 is produced mainly by thermophilic fungi. Several experiments did not reveal any other specific compound produced by S. thermophilum that increases the hyphal extension rate of the mushroom mycelium.

Oestradiol, a new hapten for detecting nucleic acid sequences by FISH.

Oestradiol has been conjugated to allylamine-dUTP with an 11-atom spacer to allow enzymatic incorporation of the label into DNA sequences. In a comparative DNA and mRNA FISH study we have used DNA probes that were either labelled with digoxigenin, biotin or oestradiol. Results show that oestradiol-labelled probes can detect DNA and RNA sequences in FISH equally well as digoxigenin- and biotin-labelled probes. Further, no crossreactivity between the various hapten-specific antibodies and the three haptens were observed. Binding of the rabbit anti-oestradiol antibody to endogenous oestrogen in various tissues was not observed under the conditions tested. In view of the increasing demands for multi-colour DNA and mRNA FISH applications, oestradiol is a welcome addition to the collection of haptens employed in FISH.