Ten years of barcoding at the African Centre for DNA Barcoding.

The African Centre for DNA Barcoding (ACDB) was established in 2005 as part of a global initiative to accurately and rapidly survey biodiversity using short DNA sequences. The mitochondrial cytochrome c oxidase 1 gene (CO1) was rapidly adopted as the de facto barcode for animals. Following the evaluation of several candidate loci for plants, the Plant Working Group of the Consortium for the Barcoding of Life in 2009 recommended that two plastid genes, rbcLa and matK, be adopted as core DNA barcodes for terrestrial plants. To date, numerous studies continue to test the discriminatory power of these markers across various plant lineages. Over the past decade, we at the ACDB have used these core DNA barcodes to generate a barcode library for southern Africa. To date, the ACDB has contributed more than 21 000 plant barcodes and over 3000 CO1 barcodes for animals to the Barcode of Life Database (BOLD). Building upon this effort, we at the ACDB have addressed questions related to community assembly, biogeography, phylogenetic diversification, and invasion biology. Collectively, our work demonstrates the diverse applications of DNA barcoding in ecology, systematics, evolutionary biology, and conservation.

Exposing the illegal trade in cycad species (Cycadophyta: Encephalartos) at two traditional medicine markets in South Africa using DNA barcoding.

Species in the cycad genus Encephalartos are listed in CITES Appendix I and as Threatened or Protected Species in terms of South Africa's National Environmental Management: Biodiversity Act (NEM:BA) of 2004. Despite regulations, illegal plant harvesting for medicinal trade has continued in South Africa and resulted in declines in cycad populations and even complete loss of sub-populations. Encephalartos is traded at traditional medicine markets in South Africa in the form of bark strips and stem sections; thus, determining the species traded presents a major challenge due to a lack of characteristic plant parts. Here, a case study is presented on the use of DNA barcoding to identify cycads sold at the Faraday and Warwick traditional medicine markets in Johannesburg and Durban, respectively. Market samples were sequenced for the core DNA barcodes (rbcLa and matK) as well as two additional regions: nrITS and trnH-psbA. The barcoding database for cycads at the University of Johannesburg was utilized to assign query samples to known species. Three approaches were followed: tree-based, similarity-based, and character-based (BRONX) methods. Market samples identified were Encephalartos ferox (Near Threatened), Encephalartos lebomboensis (Endangered), Encephalartos natalensis (Near Threatened), Encephalartos senticosus (Vulnerable), and Encephalartos villosus (Least Concern). Results from this study are crucial for making appropriate assessments and decisions on how to manage these markets.

Visualization of freezing damage.

Freeze-cleaving can be used as a direct probe to examine the ultrastructural alterations of biological material due to freezing. We examined the thesis that at least two factors, which are oppositely dependent upon cooling velocity, determine the survival of cells subjected to freezing. According to this thesis, when cells are cooled at rates exceeding a critical velocity, a decrease in viability is caused by the presence of intracellular ice; but cells cooled at rates less than this critical velocity do not contain appreciable amounts of intracellular ice and are killed by prolonged exposure to a solution that is altered by the presence of ice. As a test of this hypothesis, we examined freeze-fractured replicas of the yeast Saccharomyces cerevisiae after suspensions had been cooled at rates ranging from 1.8 to 75,000 degrees C/min. Some of the frozen samples were cleaved and replicated immediately in order to minimize artifacts due to sample handling. Other samples were deeply etched or were rewarmed to -20 degrees C and recooled before replication. Yeast cells cooled at or above the rate necessary to preserve maximal viability ( approximately 7 degrees C/min) contained intracellular ice, whereas cells cooled below this rate showed no evidence of intracellular ice.

Association of microfilament bundles with lysosomes in polymorphonuclear leukocytes.

The juxtaposition of microfilament bundles and lysosomes seen both in thin-sectioned cells in the transmission electron microscope and in cryofractured cells in the scanning electron microscope, and the presence of short filamentous structures between lysosomes and microfilament bundles, suggest that microfilaments may be attached to lysosomal membranes and that these filaments may be involved in lysosomal movements. Further work is in progress to test these hypotheses.

Phagocytosis of bacteria by polymorphonuclear leukocytes. A freeze-fracture, scanning electron microscope, and thin-section investigation of membrane structure.

The changes in membrane structure of rabbit polymorphonuclear (PMN) leukocytes during bacterial phagocytosis was investigated with scanning electron microscope (SEM), thin-section, and freeze-fracture techniques. SEM observations of bacterial attachment sites showed the involvement of limited areas of PMN membrane surface (0.01-0.25mum(2)). Frequently, these areas of attachment were located on membrane extensions. The membrane extensions were present before, during, and after the engulfment of bacteria, but were diminished in size after bacterial engulfment. In general, the results obtained with SEM and thin-section techniques aided in the interpretation of the three-dimensional freeze-fracture replicas. Freeze-fracture results revealed the PMN leukocytes had two fracture faces as determined by the relative density of intramembranous particles (IMP). Membranous extensions of the plasma membrane, lysosomes, and phagocytic vacuoles contained IMP's with a distribution and density similar to those of the plasma membrane. During phagocytosis, IMPs within the plasma membrane did not undergo a massive aggregation. In fact, structural changes within the membranes were infrequent and localized to regions such as the attachment sites of bacteria, the fusion sites on the plasma membrane, and small scale changes in the phagocytic vacuole membrane during membrane fusion. During the formation of the phagocytic vacuole, the IMPs of the plasma membrane appeared to move in with the lipid bilayer while maintaining a distribution and density of IMPs similar to those of the plasma membranes. Occasionally, IMPs were aligned to linear arrays within phagocytic vacuole membranes. This alignment might be due to an interaction with linearly arranged motile structures on the side of the phagocytic vacuole membranes. IMP-free regions were observed after fusion of lysosomes with the phagocytic vacuoles or plasma membrane. These IMP-free areas probably represent sites where membrane fusion occurred between lysosomal membrane and phagocytic vacuole membrane or plasma membrane. Highly symmetrical patterns of IMPs were not observed during lysosomal membrane fusion.

Propylthiouracil-induced hepatic damage.

Two cases of propylthiouracil-induced liver damage have been observed. The first case is of an acute type of damage, proven by rechallenge; the second presents a clinical and histologic picture resembling chronic active hepatitis, with spontaneous remission.

Intramembranous particles in erythrocyte, reticulocyte and erythroblastic leukemic cells of the rat: a model system for erythrocyte maturation.

The density and size distribution of intramembranous particles (IMP) were determined for cells of the erythroid series. The number and size of IMP were measured on both fracture faces of erythroblastic leukemia cells, phenylhydrazine-induced reticulocytes and mature erythrocytes. We found that the number of IMP adhering to the protoplasmic fracture face of the plasma membrane increased with increasing maturation, while the number of particles adhering to the external fracture face did not correlate with maturational stage. In general, the mean size of particles adhering to both fracture faces decreased with increasing maturation after the erythroblastic stage. We interpret these results to mean that the IMP seen are derived from more than one macromolecular species and that they are distributed asymmetrically in the plasma membrane.

Human marrow erythropoiesis in culture: III. Ultrastructural and cytochemical studies of cellular interactions.

Human erythroblasts cultured with a methylcellulose clonal assay technique were studied with ultrastructural and cytochemical methods. The intact colonies contained only erythroblasts at a similar stage of maturation, and no macrophages were identified within the colonies. The cultured erythroblasts demonstrated many of the morphologic features described in vivo. Neutral and acid glycoconjugates identified on the plasmalemma stained in a similar way to that seen in vivo with a concanavalin A horseradish peroxidase bridge and dialyzed iron technique. Weak acid phosphatase activity and ferritin-like particles were demonstrated in siderosomes, but these structures lacked peroxidase activity and dialyzed iron reactive acid mucosubstance. Transmission and scanning electron microscopy identified numerous processes which connected early erythroblasts and resembled those described in the marrow of patients with dyserythropoietic disorders. These findings suggest the presence of abnormalities in cultured erythrocytes which should be considered when evaluating pathologic specimens in vitro.

Streptococcal pancarditis.

An unusual association of pancarditis due to Streptococcus viridans is described. The pathologic findings appear to indicate that the myocardial infection and pericarditis were blood borne.

Isozyme and allozyme markers distinguishing two morphologically similar, medically important Mastomys species (Rodentia: Muridae).

BACKGROUND: Two common southern African mice species, Mastomys coucha and M. natalensis, are widely distributed throughout the subregion and overlap in many areas. They also share a high degree of morphological similarity, making them impossible to distinguish in the field at present. These multimammate mice are documented carriers of serious disease vectors causing Lassa fever, plague and encephalomyocarditis, which coupled to their cohabitation with humans in many areas, could pose a significant health risk. A preliminary study reported the presence of isozyme markers at three loci (GPI-2, PT-2, -3) in one population each of M. coucha and M. natalensis. Two additional populations (from the Vaal Dam and Richards Bay) were sampled to determine the reliability of these markers, and to seek additional genetic markers. RESULTS: Fifteen proteins or enzymes provided interpretable results at a total of 39 loci. Additional fixed allele differences between the species were detected at AAT-1, ADH, EST-1, PGD-1, Hb-1 and -2. Average heterozygosities for M. coucha and M. natalensis were calculated as 0.018 and 0.032 respectively, with a mean genetic distance between the species of 0.26. CONCLUSIONS: The confirmation of the isozyme and the detection of the additional allozyme markers are important contributions to the identification of these two medical and agricultural pest species.

Scanning electron microscopy of Dalkon Shield tails.

Scanning electron micrographs of Dalkon Shield tails removed from asymptomatic patients show a variety of microbes and debris throughout their entire length. Apparently, even in undamaged tails, bacterial flora thrive in the protein-rich environment within the multifilament tail. The presence of microbes in the portion of the tail beyond the double knot indicates that an alternative mechanism of microbial transport can occur. Since transient endometritis often occurs immediately after insertion of intrauterine devices, microbes may come in contact with both exposed ends of the multifilament tail and be drawn into the tail by capillary action from the uterine environment down the tail toward the double knot as well as upward from the vagina. Such microorganisms could serve as an inoculum for infection.

Scanning electron microscopy of copper-containing intrauterine devices: long-term changes in utero.

The addition of metallic copper wire to a polyethylene intrauterine device dramatically increases the efficacy of that device. A series of copper-containing Tatum-T devices which had been carried in utero for 2 to 3 years was examined by scanning electron microscopy. Few of these devices showed evidence of corrosion or pitting of the copper, as had been reported previously. Almost all of the Ts showed a definite pattern of layering of fibrinoid deposits; intact cells, cellular debris, and crystalline structures were also seen. The micrographs show an extensive buildup of surface layers which, we believe, encase the copper in a semipermeable matrix. This matrix may retard the diffusion of copper compounds into the uterus sufficiently to compromise the contraceptive action of the copper.

Preparation of fingernails for trace element analysis.

There are substantial differences in the reported elemental composition of human nails. Most investigators have used extensive washing procedures to minimize environmental contamination, however, such washing poses the risk of extraction of elements bound to the nail matrix. To determine if a portion of this variability could be accounted for by the "washing solutions" used by different investigators, nails were washed in nine solvents previously used for cleaning nails and their residual elemental composition measured by atomic absorption spectroscopy or energy dispersion analysis. In general, treatment with organic solvents resulted in less elemental loss than did treatment with aqueous detergents, while aqueous acids caused the greatest loss. Organic solvents more readily extracted iron and magnesium than calcium, copper and zinc. Virtually all of the magnesium was extracted by distilled water or aqueous detergents.

Fluid migration through the tail of the Dalkon shield intrauterine device.

X-ray radiographs were used to monitor the migration of Renouve -dip radiopaque tracer through the multifilament tail of Dalkon Shield intrauterine devices (IUDs). The movement of the tracer was more rapid and migrated a greater distance through the long end of the tail than through the short end of the tail. The double knot serves as at least a temporary barrier to the migration of tracer from the long end of the tail, but this barrier was circumvented when drops of Renouve -dip radiotracer were placed at both ends of the tail. Migration of Renouve -dip radiotracer was prevented when the end of the Dalkon Shield tail was fused by heating. No migration of this tracer occurred on the tails of commonly used IUDs which possessed monofilament tails, thus demonstrating that fluid migration occurs within and not on the surface of the multifilament tail.

PIP: X-ray radiographs were used to monitor the migration of Renouve-dip radiopaque tracer through the multifilament tail of the Daldon Shield IUD. The movement of the tracer was more rapid and migrated a greater distance through the long end of the tail than through the short end. The double knot serves as at least a temporary barrier to the migration of the tracer from the long end of the tail, but this barrier was circumvented when drops of Renouve-dip radiotracer were placed at both ends of the tail. Migration of Renouve-dip radiotracer was prevented when the end of the Dalkon Shield tail was fused by heating. No migration of this tracer occurred on the tails of commonly used IUDs which possessed monofilament tails, thus demonstrating that fluid migration occurs within and not on the surface of the multifilament tail.

Freeze-dried microarterial replacement allografts in rabbits.

Microvascular techniques offer important alternatives for reconstructive head and neck surgery. To test the viability of freeze-dried allografts, a pilot experimental study was performed using the rabbit model. Freeze-dried preserved arterial allografts were implanted into femoral artery defects in eight subjects. After 6 weeks, all grafts were harvested and prepared for histological and electron microscopic analysis. Immediate patency was 100%. One subject was excluded on the third postoperative day. Of the seven remaining grafts, three (43%) were patent at 6 weeks. These results are comparable to previous data obtained using freeze-dried arterial allografts in the same animal model. Although further investigation is required, this pilot study suggests possible future application of cryopreserved vascular micrografts.

Evidence for an active immune response in acute hyperthyroidism (Graves' disease).

Activated lymphocytes, identified by an autoradiographic labeling method, were found to be present in the peripheral blood of the majority of 20 patients with acute hyperthyroidism (Graves' disease). The number of such cells in the blood was significantly greater than that found in 30 healthy controls (p less than 0.00001), and in 13 patients who had previously suffered from acute hyperthyroidism, and who were judged to be euthyroid following therapy (p less than 0.025). This latter group included two patients in whom such activated lymphocytes had been found in the blood during the acute phase of their illness. Furthermore, there were significant differences between the number of such activated circulating lymphocytes in the group of patients with acute hyperthyroidism and five patients suffering from hyperthyroidism due to a toxic thyroid nodule (p less than 0.001), five patients suffering from primary myxedema (p less than 0.001), or in 14 patients with a nontoxic multinodular goiter (p less than 0.05). Identification and counting of circulating T and B lymphocytes by fluorescent immunolabeling and rosette-forming techniques in a small number of the patients with acute hyperthyroidism failed to reveal significant differences from the normal. The results suggest that in acute hyperthyroidism there is active stimulation of the cellular immune system, and that this effect is specific to the early, untreated phase of the disease. This response is different to other thyroid diseases, including hyperthyroidism due to a toxic thyroid nodule.

Myo-inositol clearance in renal failure and in patients with normal kidney function.

In order to determine the mechanism of elevated serum inositol in renal failure, the clearance values of inositol and of creatinine were measured in patients with normal kidney function and in those whose renal function was impaired due to varying causes. Mean serum inositol level in controls was 5.6 microgram/ml, and in patients with renal failure 28.6 microgram/ml. In control patients, inositol clearance was 2.8 ml/min, and tubular reabsorption of inositol was found to be over 97 percent. The inositol clearance of patients in renal failure varied from 0.62 to 17 ml/min. The ratio inositol clearance/creatinine clearance was elevated in uremic patients. Total amounts of inositol excreted in the urine of uremic patients were consistently higher than those excreted by control patients. The elevated serum inositol levels seen in renal failure were therefore not primarily caused by inability of the diseased kidney to excrete inositol.

Videoanalysis of chemokinesis: characterization of speed, persistence and orientation in an agarose assay.

Time-lapse video recording and off-line computer analysis were used to characterize the chemokinetic behavior of individual human neutrophils migrating in an agarose assay. When neutrophils were stimulated with an isotropic concentration of formyl-methionyl-leucyl-phenylalanine (fMLP), they migrated with a mean speed of 9.6 micron per min and oriented at random. The ratio of net displacement to total distance travelled (persistence of locomotion) was 0.66, indicating that neutrophils maintained some directional persistence even in the absence of a gradient of fMLP. The speed and persistence of locomotion index were correlated because both faster and slower cells had high persistence, while only slower cells had low persistence. The orientation angle was independent of both speed and persistence of locomotion. These are the first reported direct measurements of the chemokinetic locomotion of neutrophils using the agarose assay.

Kinematic analysis of chemotaxis of fresh and stored neutrophils.

When neutrophils are isolated from the circulation the first function to begin to deteriorate is chemotaxis. To characterize the loss of chemotaxis that occurs during storage, a computer-assisted video motion analysis of neutrophils responding to formyl-methionyl-leucyl-phenylalanine (FMLP) was used in an agarose assay. The chemotactic speed, velocity, and orientation angle were measured, and a persistence of locomotion index (velocity/speed) and chemotropic index (cosine of the orientation angle) were calculated for fresh neutrophils and neutrophils stored in plasma at 20 to 22 degrees C for 24 hours. The data reveal that: (1) the frequency distribution of speed for individual stored cells had a different shape than that of fresh cells owing to a subpopulation of stored cells (approximately 35 percent) which migrated at a slower mean speed; (2) the frequency distribution of orientation for fresh cells is not normally distributed and contains a subpopulation (approximately nine percent of the total) of cells which orient at random in a gradient; (3) the precision of orientation of the majority of stored cells is comparable to that of fresh cells, but approximately 35 percent of the stored cells orient at random in a chemoattractant gradient; (4) neither the persistence index nor the orientation of both fresh and stored cells were correlated with speed; (5) the chemotropic index and persistence index are correlated, and this correlation is not altered by storage suggesting that stored cells which show decreased persistence also show a decreased chemotropic index. It is proposed that neutrophils respond to a gradient of fMLP with either fast, persistent, accurately oriented locomotion or slower, less persistent, randomly oriented locomotion. In addition to those neutrophils which do not migrate in response to fMLP, it is proposed that there are two subpopulations of motile neutrophils. Storage at 20 to 22 degrees C induces shifts between these three modes of behavior.

Allozyme variation in four populations of African whitebacked vultures (Gyps africanus) and phylogenetic relationships between four vulture species from southern Africa.

Genetic variation detected by protein electrophoresis at 41 presumptive gene loci was assayed in four populations of Gyps africanus and compared to values previously obtained for Gyps coprotheres. Values calculated for percentage of polymorphic loci (P=34.15%, 0.99 criterion) and average heterozygosity (&Hmacr;=0.108, +/-0.032) in G. africanus, confirm low levels of genetic variation as reported for G. coprotheres. Allele frequency data, assessed at 19 loci, were obtained to evaluate genetic differentiation among four vulture species. Six (31.58%) of the 19 shared loci were polymorphic. Values of 1.26 (+/-0.1), 26.32% and 0.076 (+/-0.047) for G. africanus, 1.21 (+/-0.1), 21.05% and 0.097 (+/-0.045) for Torgos tracheliotus, 1.11 (+/-0.7), 21.05% and 0.053 (+/-0.053) for Neophron percnopterus and 1.05 (+/-0.5), 5.26% and 0.044 (+/-0.047) for G. coprotheres were obtained for the mean number of alleles per locus, P and &Hmacr;, respectively. An average between-population fixation index (F(ST)) value of 0.322 was obtained, which is indicative of significant (P<0.01) differentiation between the four accipitrid species studied. Considerable concordance was obtained between dendograms produced from different analyses, pointing to the distinctiveness of N. percnopterus, which has evolved along a separate lineage as G. africanus, G. coprotheres and T. tracheliotus. Along the latter lineage G. africanus is clustered together with G. coprotheres which is consistent with the morphological similarities of these species.