Mutation enrichment in human DNA samples via UV-mediated cross-linking

Detection of low-level DNA mutations can reveal recurrent, hotspot genetic changes of clinical relevance to cancer, prenatal diagnostics, organ transplantation or infectious diseases. However, the high excess of wild-type (WT) alleles, which are concurrently present, often hinders identification of salient genetic changes. Here, we introduce UV-mediated cross-linking minor allele enrichment (UVME), a novel approach that incorporates ultraviolet irradiation (∼365 nm UV) DNA cross-linking either before or during PCR amplification. Oligonucleotide probes matching the WT target sequence and incorporating a UV-sensitive 3-cyanovinylcarbazole nucleoside modification are employed for cross-linking WT DNA. Mismatches formed with mutated alleles reduce DNA binding and UV-mediated cross-linking and favor mutated DNA amplification. UV can be applied before PCR and/or at any stage during PCR to selectively block WT DNA amplification and enable identification of traces of mutated alleles. This enables a single-tube PCR reaction directly from genomic DNA combining optimal pre-amplification of mutated alleles, which then switches to UV-mediated mutation enrichment-based DNA target amplification. UVME cross-linking enables enrichment of mutated KRAS and p53 alleles, which can be screened directly via Sanger sequencing, high-resolution melting, TaqMan genotyping or digital PCR, resulting in the detection of mutation allelic frequencies of 0.001–0.1% depending on the endpoint detection method. UV-mediated mutation enrichment provides new potential for mutation enrichment in diverse clinical samples.     

A mathematical model for fluid shear-sensitive 3D tissue construct development

This research studies dynamic culture for 3D tissue construct development with computational fluid dynamics. It proposes a mathematical model to evaluate the impact of flow rates and flow shear stress on cell growth in 3D constructs under perfusion. The modeling results show that dynamic flow, even at flow rate as low as 0.002 cm/s, can support much better mass exchange, higher cell number, and more even cell and nutrient distribution compared to static culture. Higher flow rate can further improve nutrient supply and mass exchange in the construct, promoting better nutritious environment and cell proliferation compared to lower flow rate. In addition, consideration of flow shear stress predicts much higher cell number in the construct compared to that without shear consideration. While the nutrient can dominate shear stress in influencing cell proliferation, the shear effect increases with flow rate. The proposed model helps tissue engineers better understand the cell-flow relationship at the molecular level during dynamic culture.     

Circulating levels of α‐tocopherol and retinol in free‐ranging African elephants (Loxodonta africana)

To date, there are no detailed reports of circulating levels of plasma α‐tocopherol and retinol for large samples of free‐ranging African elephants (Loxodonta africana). This survey study measured natural circulating levels of α‐tocopherol as a measure of vitamin E activity and retinol as an indicator of vitamin A activity, in 70 free‐ranging African elephants captured at Kruger National Park as part of a translocation program. Mean levels of α‐tocopherol and retinol were found to be 0.613 ± 0.271 μg/mL and 0.039± 0.007 μg/mL, respectively, and did not vary significantly across sex or age class. Elephants appear to normally have low circulating levels of both these nutrients compared with domestic herbivore species; values from healthy, free‐ranging elephant populations may provide useful data for assessing nutrient status of captive animals. Zoo Biol 18:319–323, 1999.© 1999 Wiley‐Liss, Inc.     

Prediction of non-linear pharmacokinetics in humans of an antibody-drug conjugate (ADC) when evaluation of higher doses in animals is limited by tolerability: Case study with an anti-CD33 ADC

For antibody-drug conjugates (ADCs) that carry a cytotoxic drug, doses that can be administered in preclinical studies are typically limited by tolerability, leading to a narrow dose range that can be tested. For molecules with non-linear pharmacokinetics (PK), this limited dose range may be insufficient to fully characterize the PK of the ADC and limits translation to humans. Mathematical PK models are frequently used for molecule selection during preclinical drug development and for translational predictions to guide clinical study design. Here, we present a practical approach that uses limited PK and receptor occupancy (RO) data of the corresponding unconjugated antibody to predict ADC PK when conjugation does not alter the non-specific clearance or the antibody-target interaction. We used a 2-compartment model incorporating non-specific and specific (target mediated) clearances, where the latter is a function of RO, to describe the PK of anti-CD33 ADC with dose-limiting neutropenia in cynomolgus monkeys. We tested our model by comparing PK predictions based on the unconjugated antibody to observed ADC PK data that was not utilized for model development. Prospective prediction of human PK was performed by incorporating in vitro binding affinity differences between species for varying levels of CD33 target expression. Additionally, this approach was used to predict human PK of other previously tested anti-CD33 molecules with published clinical data. The findings showed that, for a cytotoxic ADC with non-linear PK and limited preclinical PK data, incorporating RO in the PK model and using data from the corresponding unconjugated antibody at higher doses allowed the identification of parameters to characterize monkey PK and enabled human PK predictions.     

Fragments or propagules? Reproductive tradeoffs among Callyspongia spp. from Florida coral reefs

Fragmentation and propagule formation are alternative reproductive strategies found in both plants and animals, with the latter generally providing greater dispersal capability. When both strategies occur, life history theory predicts that resources should be divided between the two. On coral reefs, both strategies are exhibited by branching corals and sponges, which are broken‐up after storm events and rapidly recolonize. In this study, we compared two congeneric Caribbean reef sponges, Callyspongia armigera, which is branched and easily fragmented, and C. vaginalis, which is not, to test whether there is a tradeoff in growth and propagule formation for C. armigera relative to C. vaginalis. Both species were equally abundant on coral reefs off Key Largo, Florida (10.1 ± 3.7 vs 11.9 ± 3.0 per 100 m², respectively), suggesting that they are equally successful relative to two other non‐fragmenting congeneric species (C. fallax and C. plicifera) that are much less common. The number of substratum attachment points per sponge was significantly higher for C. armigera compared to C. vaginalis (2.31 ± 1.47 vs 1.03 ± 0.18 sponge^?1), providing further evidence of the reliance of C. armigera on fragmentation, and of C. vaginalis on recruitment from larval settlement and subsequent growth. Growth rates in predator‐exclusion experiments were ～4‐fold higher for C. armigera compared to C. vaginalis (0.36 ± 0.31 vs 0.08 ± 0.11 % initial mass day^?1), but C. armigera produced ～13‐fold fewer propagules than C. vaginalis (0.04 ± 0.22 vs 0.53 ± 1.08 % tissue area). Our results support a tradeoff between growth and propagule output for C. armigera relative to C. vaginalis, suggesting that these closely related sponge species took different evolutionary trajectories in reconciling their resource constraints.     

Production of deferriferrioxamines B and E from a ferroverdin-producing Streptomyces species.

When Streptomyces sp. strain Wak. A-305 is cultured in iron (III)- or cobalt (II)-containing media, tris(p-vinylphenyl-4-hydroxy-3-nitrosobenzoato-N3, O4)ferrate(II) (ferroverdin) or the analogous tris-cobalt(III) complex, respectively, is produced intracellularly. In contrast, when cultured in transition metal ion-limiting media. Streptomyces sp. strain Wak. A-305 did not produce p-vinylphenyl 4-hydroxy-3-nitrosobenzoate (deferroferroverdin). Instead, deferriferrioxamines B and E, which have been characterized from other streptomycetes, were isolated extracellularly and may act as siderophores in this organism.U     

Therapeutic abortion with concurrent sterilization: comparison of methods

Among patients seeking abortion are a group who request concurrent sterilization. This study compared three combinations of abortion and sterilization techniques. Suction termination with laparoscopic tubal ligation appears to be the combination of choice, for the mean hospital stay of patients undergoing this procedure was two days and the operative morbidity was 3.1%. For suction termination with tubal ligation at laparotomy, and hysterotomy with tubal ligation the mean hospital stays were 6 and 5.7 days respectively and the operative morbidity was four to five times greater.     

Separation of Antioxidant-Rich Alternanthera Sessilis Red Extracts by Sephadex LH-20 and Identification of Polyphenols Using HPLC-QToF-MS/MS

This study aimed to fractionate Alternanthera sessilis Red (ASR) crude extracts and determine their antioxidant activities as well as the related active components in the whole plant. ASR was extracted with water and ethanol, and further separated using a Sephadex LH-20 column. Following the assessments of the polyphenolic contents and antioxidant activities of crude extracts (H₂O_ASR and EtOH_ASR) and fractions, a HPLC-QToF analysis was performed on the crude extracts and selected fractions (H₂O_ASR FII and EtOH_ASR FII). Three water fractions (H₂O_ASR FI, FII and FIII) and four ethanolic fractions (EtOH_ASR FI, FII, FIII and FIV) were derived from their crude extracts, respectively. EtOH_ASR FII exhibited the greatest total phenolic content (120.41 mg GAE/g fraction), total flavonoid content (223.07 mg RE/g fraction), and antioxidant activities (DPPH IC₅₀=159.43 μg/mL; FRAP=1.93 mmol Fe²⁺/g fraction; TEAC=0.90 mmol TE/g fraction). Correlation analysis showed significant (p<0.01) positive correlations between both TPC (r=0.748–0.970) and TFC (r=0.686–0.949) with antioxidant activities in the crude extracts and fractions. Flavonoids were the major compounds in the four selected samples tentatively identified using HPLC-QToF-MS/MS, with the highest number of 30 polyphenol compounds detected in the most active fraction, EtOH_ASR FII.     

Sensory,motor, and postganglionic sympathetic neurons forming the ulnar and radial nerves of two macaque species: Macaca nemestrina and Macaca fascicularis

The motor, sensory, and postganglionic sympathetic neurons forming the left ulnar and right radial nerves of long-tailed macaques (Macaca fascicularis) and pigtailed macaques (Macaca nemestrina) were localized by the horseradish peroxidase method of tracing neuronal connections. The ulnar and radial motoneurons formed a longitudinal column of variable extent in the lateral part of the ventral horn. In most animals, the ulnar motoneurons extended between the caudal ends of the C7 and T1 segments; the radial motoneurons extended between the rostral level of the C4 and the middle part of T1 segments. Although there were areas of overlap in the spinal distribution of ulnar and radial motoneurons, the ulnar motoneurons were located more dorsally and dorsolaterally than were the radial motoneurons. In most animals, labelled sensory neurons whose axons run with the ulnar nerve occurred in the C8–T4 dorsal root ganglia, and those whose axons run with the radial nerve occurred in the C5–T3 ganglia. The radial sympathetic neurons were distributed in stellate through T7 paravertebral sympathetic ganglia, and the ulnar sympathetic neurons were distributed in stellate through T4 paravertebral sympathetic ganglia. Though the motor, sensory, and sympathetic neurons forming the ulnar and radial nerves had wide segmental distributions, all showed peak frequencies in two segments. The cross-sectional areas of the motor, sensory, and postganglionic sympathetic neurons forming the radial and ulnar nerves were measured in the animal that showed the greatest amount of labelling for each nerve. The ulnar and radial motoneurons had a similar range of sizes, with cross-sectional areas between 120 and 2,160 μm². Most were smaller than 900 μm². The sensory neurons forming the ulnar and radial nerves also displayed a similar range of sizes, measuring between 120 and 3,360 μm² in cross-sectional area. Most neurons measured between 201 and 800 μm². The ulnar sympathetic neurons measured between 120 and 840 μm², and the radial neurons between 120 and 2,120 μm². In both cases, most neurons measured between 120 and 600 μm². The mean cross-sectional area for the radial sympathetic neurons was, however, larger than that for the ulnar sympathetic neurons.