Optimization of the Magnetic Recovery of Hits from One-Bead-One-Compound Library Screens.

On-bead screening of one-bead-one-compound (OBOC) libraries is a useful procedure for the identification of protein ligands. An important aspect of this experiment is the method by which beads that bind the target protein are separated from those that do not. Ideally, such a method would be rapid and convenient and result in the isolation of 100% of the "hits" with no false positives (beads that display compounds that are not good ligands for the target). We introduced a technique in which beads that have bound a labeled target protein can be magnetized, thus allowing their convenient isolation ( Astle et al. Chem. Biol. 2010 , 17 , 38 - 45 ). However, recent work in our laboratory and others has shown that magnetic hit recovery can result in the isolation of large numbers of false positives and has also suggested that many true hit beads are missed. In this study, we employ a well-defined model system to examine the efficiency of various magnetic hit isolation protocols. We show that the choice of reagents and the particular operations employed are critical for optimal results.

The performance of Orma Boran and Maasai Zebu crossbreeds in a trypanosomosis endemic area of Nguruman, south western Kenya.

Studies on the trypanotolerance of Orma Boran X Maasai Zebu (Orma Zebu) crossbred cattle (F1 progeny) and pure-bred Maasai Zebu contemporaries were carried out in Nguruman, south western Kenya. The two groups were monitored from birth for a period of 2 years. The incidence of trypanosomosis, parasitaemia, packed cell volume (PCV), body mass and average daily mass gain were monitored. During the study period, overall trypanosomosis incidence was low (3%). The crossbred cattle had a higher incidence of infection (61% vs 39%). The mean PCV and mean mass gain for the crossbred cattle was higher than that of the Maasai Zebu. The mean calf body mass at weaning (8 months) for the Orma Zebu and Maasai Zebu was 72 kg and 64 kg, respectively, while at 18 months of age their mean body mass was 164 kg and 123 kg, respectively. During the rainy season significant differences in average daily mass gains were noted (P < 0.05). The superior mass gain of the Orma Zebu observed during the rainy season, despite higher infection rates, indicate an enhanced trypanotolerance. Moreover, the better performance of the Orma Zebu is an attribute that could be exploited in the adoption of the trypanotolerance genotype, as a sustainable trypanosomosis control strategy.

Detection of Trypanosoma brucei rhodesiense in animals from sleeping sickness foci in East Africa using the serum resistance associated (SRA) gene.

The human serum resistance associated (SRA) gene has been found exclusively in Trypanosoma brucei rhodesiense, allowing the unequivocal detection of this pathogen in reservoir hosts and the tsetse vector without recourse to laborious strain characterisation procedures. We investigated the presence of the SRA gene in 264 T. brucei ssp. isolates from humans, domestic animals and Glossina pallidipes from foci of human trypanosomiasis in Kenya and Uganda. The SRA gene was present in all isolates that were resistant to human serum, and absent from all serum sensitive isolates tested. Further, the gene was present in all isolates that had previously been shown to be identical to human infective trypanosomes by isoenzyme characterisation. The SRA gene was detected in isolates from cattle, sheep, pigs, dog, reedbuck, hyena and G. pallidipes from sleeping sickness foci, but was not found in Trypanosoma evansi or in Trypanosoma brucei gambiense isolates. The present study indicates that the SRA gene may be invaluable in detecting and differentiating T. brucei rhodesiense from other T. brucei ssp. in reservoir hosts and tsetse.

Identification of trypanosomes in Glossina pallidipes and G. longipennis in Kenya.

The polymerase chain reaction (PCR) was used to identify trypanosomes in Glossina pallidipes and G. longipennis caught in Kenya. Of 3826 flies dissected, 188 (4.9%) were parasitologically positive overall. The infection rate in G. pallidipes was 5.7% (187 of 3301 flies), but only one of 525 G. longipennis was infected (infection rate 0.2%). There was a higher infection rate in female G. pallidipes flies than male flies (chi(2) = 18.5, P < 0.001) and odds ratio = 2.5 (95% 1.6, 3.7). The infected flies were analysed by PCR using 10 sets of primers specific for species and subgroups within the subgenera Nannomonas, Trypanozoon and Duttonella. Of 188 parasitologically positive samples, PCR identified 137 (72.9%), leaving 51 (27.1%) non-identified. We recorded infection rates of 47.2% for Trypanosoma congolense savannah, forest and kilifi subgroups, 20.9% for T. simiae/T. simiae tsavo/T. godfreyi, 14.9% for T. brucei ssp. and 13.8% for T. vivax. Thirty-nine (26.7%) flies had mixed infections, with a minor association between T. congolense savannah/T. simiae tsavo/T. godfreyi (chi(2) = 6.93, d.f. = 1, P < 0.05). The relative proportion of each trypanosome species or subgroup varied between fly belts with T. congolense (all subgroups) being the most abundant and T. godfreyi the least. Statistical analysis showed that dissection method and PCR test classified infections independently (chi(2) = 10.5, d.f. = 1, P < 0.05 and kappa = 0.38). This study shows that pathogenic trypanosomes are widespread in all sampled testes fly belts with G. pallidipes as the main vector. Further, PCR test is more reliable in detecting and identifying trypanosomes than dissection method.

Blunt abdominal trauma in children at Kenyatta National Hospital.

OBJECTIVE: To determine the pattern of intra-abdominal injuries arising from blunt abdominal trauma and evaluate the management of blunt abdominal trauma and its outcome. DESIGN: Retrospective study. SETTING: Paediatric Surgical Department, Kenyatta National Hospital (KNH), Nairobi. SUBJECTS: Fifty-five children who were admitted and treated for blunt abdominal trauma in KNH between January 1983 and December 1993. MAIN OUTCOME MEASURES: Morbidity as determined by operative management and complications, mortality and period of hospital stay. RESULTS: Incidence of trauma was high in males with male to female ratio of 2:1. Incidence of trauma was prevalent in seven year age group. Motor vehicle accidents accounted for the majority of injuries. Most patients arrived late in hospital. Sixty per cent underwent laparotomy. Three quarters of these had positive findings. The spleen was the organ most commonly injured. Hypovolaemic shock was the most frequent complication. One patient died during management. On average, patients stayed for seven days in the ward before discharge. CONCLUSION: Blunt abdominal trauma more commonly affected male children. Motor vehicle accidents were a major aetiologic factor in blunt abdominal trauma in children seen in Kenyatta National Hospital. Twenty-five per cent of the children were subjected to unnecessary operation. This was due to unavailability of sensitive diagnostic modalities.

Ultrastructural studies in the hearts of dogs infected with trypanosoma brucei

In an attempt to study the mechanisms of heart damage in African trypanosomiasis; a group of dogs were infected with Trypanosoma brucei. The dogs developed an actue disease syndrome; and if not treated became terminally sick during the fourth week of infection. Post mortem and light microscopic examination demonstrated severe pancarditis involving the musculature; valves; vasculature and the conducting system. Transmission electron microscopy confirmed the histological findings and in addition revealed other changes in the heart. These included distention of myocardial cells with fluid; swelling of mitochondria and damage to their cristal membranes; and lipid deposition. Cellular infiltration; fibrin deposition; occluded capillaries and damaged autonomic nerve gagnlia were abserved. The changes in muscle cells; vasculature and nerve tissue may have been due to excessive inflammatory reactions caused bvy biologically active substances generated by trypanosomes themselves; or by immunological reactio;ns between trypanosomes and inflitrating cells. Such changes exacerbated the severity of heart damage; and possibly death due to heart failure