A cost-effective high-throughput metabarcoding approach powerful enough to genotype ~44 000 year-old rodent remains from Northern Africa.

We present a cost-effective metabarcoding approach, aMPlex Torrent, which relies on an improved multiplex PCR adapted to highly degraded DNA, combining barcoding and next-generation sequencing to simultaneously analyse many heterogeneous samples. We demonstrate the strength of these improvements by generating a phylochronology through the genotyping of ancient rodent remains from a Moroccan cave whose stratigraphy covers the last 120 000 years. Rodents are important for epidemiology, agronomy and ecological investigations and can act as bioindicators for human- and/or climate-induced environmental changes. Efficient and reliable genotyping of ancient rodent remains has the potential to deliver valuable phylogenetic and paleoecological information. The analysis of multiple ancient skeletal remains of very small size with poor DNA preservation, however, requires a sensitive high-throughput method to generate sufficient data. We show this approach to be particularly adapted at accessing this otherwise difficult taxonomic and genetic resource. As a highly scalable, lower cost and less labour-intensive alternative to targeted sequence capture approaches, we propose the aMPlex Torrent strategy to be a useful tool for the genetic analysis of multiple degraded samples in studies involving ecology, archaeology, conservation and evolutionary biology.

Isolation and partial characterization of a prothrombin-activating enzyme from the venom of the Australian rough-scaled snake (Tropidechis carinatus).

A prothrombin activator from the venom of Tropidechis carinatus has been isolated by means of gel filtration and benzamidine-based affinity chromatography, a novel use of the latter technique. Two bands possessing prothrombinase activity were obtained from the affinity chromatography procedure and designated A1 and A2. The bulk of the enzyme activity was recovered in peak A2 which represented 27-31% of the starting activity and a 14-16-fold purification. The venom contained, in total, around 5% by weight of the two isoforms of the prothrombin activator. The two fractions were electrophoretically similar on polyacrylamide electrophoresis, migrating with a mol. wt of 64,500 under native conditions and as a single band of 41,500 under reducing conditions. The prothrombinase was dependent on factor Va, phospholipid and calcium ions for its activity and is, thus, a member of the type II class of prothrombinases requiring such co-factors. The enzyme did not possess any phospholipase activity nor did it cleave the substrates N-alpha-benzoyl-L-arginine-p-nitroanilide (BAPNA), N-benzoyl-L-tyrosine ethyl ester (BTEE), azocollagen or azocasein, indicating a lack of amidolytic, esterolytic and broad-spectrum protease activity.

Structure of the ascarosides from Ascaris suum.

Six glycosides have been identified from the nematode Ascaris suum. The glycon part of all six glycosides is alpha L-3,6-dideoxymannose, previously known as ascarylose. The major components (4 and 5) and the minor components (8 and 9) have been shown by NMR and electrospray MS to involve a mixture of two homologous aglycons: the 2, omega-1 diols of hentriacontane and tritriacontane. Compounds 4 and 5 are glycosylated on only one of the hydroxy groups, while 8 and 9 are glycosylated on both. These compounds resemble ascarosides B and C previously isolated from Parascaris equorum. However, these aglycons are reported to be based on the 2,6-diol of hentriacontane. Compounds 6 and 7 are based on 2-hydroxy-nonacosane and 2-hydroxy-28-methylnonacosane glycosylated at C-2 with the same sugar. Although 6 and 7 are related to ascaroside A, previously isolated from P. equorum, these earlier reports suggest the chain in ascaroside A to be unbranched.

Isolation and characterisation of two haemorrhagic proteins (HTa and HTb) from the venom of Bitis gabonica (Gaboon viper).

Two distinct haemorrhagic proteinases, HTa and HTb, were isolated from the venom of Bitis gabonica by gel filtration and ion-exchange chromatography with native mol. wts of 180,000 and 111,000, respectively. After reduction with dithiothreitol, smaller mol. wts of 77,600 and 69,200 were recorded for HTa and HTb, suggesting that under native conditions the haemorrhagins exist as dimeric molecules. Both toxins possessed caseinolytic and collagenase activity although HTa was 15-36 times more potent than HTb with respect to collagenase activity. No zinc could be detected in the toxins; however, dialysis against ethylenediamine tetracetic acid (EDTA) reduced caseinolytic activity, suggesting the dependence of the latter on other metal ions. HTa and HTb had a marked effect on the intrinsic cascade coagulation mechanism (factors IX, XI and XII) but no effect on the final common coagulation pathway (factor X and prothrombin). Light and electron microscopical studies demonstrated that both HTa and HTb caused organ-specific lesions, with the lungs, diaphragm and body wall muscle being most affected. HTa caused widespread haemorrhage whilst HTb caused discrete focal lesions near the site of injection and elsewhere. However, both toxins appeared to cause capillary rupture by the separation of cells from one another and both caused cell detachment and cell death of bovine endothelial cells cultured in vitro, consonant with the massive disruption of capillaries seen in vivo.

Plasma zinc in hypertension/toxemia and other reproductive variables in adolescent pregnancy.

A study was conducted at Charity Hospital, New Orleans, among 272 adolescent pregnant women to ascertain the relationship of pregnancy outcome to plasma zinc level measured once at the time of enrollment. Regression analyses were performed on zinc status versus parameters concerning success of pregnancy corrected for gestational stage at specimen collection. Analysis of variance was performed on groups according to presence or absence of complications, with analyses of covariance used to analyze dichotomous groups. Low, though widely variable, plasma zinc levels were found (mean = 58 +/- 12.6 micrograms/dl). Zinc values differed significantly by gestational stage at collection, the regression coefficient indicating a decline of 0.07 micrograms/dl/day. Plasma zinc level correlated significantly with Hb, red blood cells, ferritin, and folic acid. As to course of pregnancy, women experiencing hypertension/toxemia were found to have significantly lower plasma zinc level. Among infants displaying congenital defects at birth those with undescended testes and metatarsus varus were delivered by mothers whose plasma zinc was well below the mean for the group. These findings indicate the need to investigate the influence of dietary patterns and zinc intake on maternal plasma zinc level and pregnancy outcome, further delineating the role of zinc in human reproduction, particularly hypertension of pregnancy.

Carboxylesterases (EC 3.1.1). Purification and titration of chicken, sheep, and horse liver carboxylesterases.

Chicken, sheep, and horse liver carboxylesterases have been purified by procedures involving ammonium sulfate fractionation, ion-exchange chromatography and gel filtration on Sephadex. The actual yields of the procedures described were as follows: chicken, 1 g from 2 kg of liver powder (chloroform-acetone); sheep, 200 mg from 400 g of powder (chloroform-acetone); horse, 230 mg from 800 g of powder (acetone). The purified enzymes are free of non-carboxyl-esterase protein as shown by gel electrophoresis, although they do contain electrophoretic variants. The equivalent weight of the chicken enzyme is 67,000 based on titration with p-nitrophenyl diethyl phosphate or bis(p-nitrophenyl) phosphate, whereas those of the sheep and horse enzymes are similar to 69,500 and similar to 70,000, respectively, based on titration with p-nitrophenyl dimethylcarbamate.