Deep sequencing of plant and animal DNA contained within traditional Chinese medicines reveals legality issues and health safety concerns

Traditional Chinese medicine (TCM) has been practiced for thousands of years, but only within the last few decades has its use become more widespread outside of Asia. Concerns continue to be raised about the efficacy, legality, and safety of many popular complementary alternative medicines, including TCMs. Ingredients of some TCMs are known to include derivatives of endangered, trade-restricted species of plants and animals, and therefore contravene the Convention on International Trade in Endangered Species (CITES) legislation. Chromatographic studies have detected the presence of heavy metals and plant toxins within some TCMs, and there are numerous cases of adverse reactions. It is in the interests of both biodiversity conservation and public safety that techniques are developed to screen medicinals like TCMs. Targeting both the p-loop region of the plastid trnL gene and the mitochondrial 16S ribosomal RNA gene, over 49,000 amplicon sequence reads were generated from 15 TCM samples presented in the form of powders, tablets, capsules, bile flakes, and herbal teas. Here we show that second-generation, high-throughput sequencing (HTS) of DNA represents an effective means to genetically audit organic ingredients within complex TCMs. Comparison of DNA sequence data to reference databases revealed the presence of 68 different plant families and included genera, such as Ephedra and Asarum, that are potentially toxic. Similarly, animal families were identified that include genera that are classified as vulnerable, endangered, or critically endangered, including Asiatic black bear (Ursus thibetanus) and Saiga antelope (Saiga tatarica). Bovidae, Cervidae, and Bufonidae DNA were also detected in many of the TCM samples and were rarely declared on the product packaging. This study demonstrates that deep sequencing via HTS is an efficient and cost-effective way to audit highly processed TCM products and will assist in monitoring their legality and safety especially when plant reference databases become better established.     

Is Audio Computer-Assisted Self-Interview (ACASI) Useful in Risk Behaviour Assessment of Female and Male Sex Workers,Mombasa, Kenya?

Background

Audio computer-assisted self-interview (ACASI) may elicit more frequent reporting of socially sensitive behaviours than face-to-face (FtF)-interview. However, no study compared responses to both methods in female and male sex workers (FSW; MSW) in Africa.

Methodology/Principal Findings

We sequentially enrolled adults recruited for an HIV-1 intervention trial into a comparative study of ACASI and FtF-interview, in a clinic near Mombasa, Kenya. Feasibility and acceptability of ACASI, and a comparative analysis of enrolment responses between ACASI and FtF on an identical risk assessment questionnaire were evaluated. In total, 139 women and 259 men, 81% of eligible cohort participants, completed both interviews. ACASI captured a higher median number of regular (2 vs. 1, p<0.001, both genders) and casual partners in the last week (3 vs. 2, p = 0.04 in women; 2 vs. 1, p<0.001 in men). Group sex (21.6 vs. 13.5%, p<0.001, in men), intravenous drug use (IDU; 10.8 vs. 2.3%, p<0.001 in men; 4.4 vs. 0%, p = 0.03 in women), and rape (8.9 vs. 3.9%, p = 0.002, in men) were reported more frequently in ACASI. A surprisingly high number of women reported in ACASI that they had paid for sex (49.3 vs. 5.8%, p<0.001). Behaviours for recruitment (i.e. anal sex, sex work, sex between males) were reported less frequently in ACASI. The majority of women (79.2%) and men (69.7%) felt that answers given in ACASI were more honest. Volunteers who were not able to take ACASI (84 men, and 37 women) mostly lacked reading skills.

Conclusions/Significance

About 1 in 5 cohort participants was not able to complete ACASI, mostly for lack of reading skills. Participants who completed ACASI were more likely to report IDU, rape, group sex, and payment for sex by women than when asked in FtF interview. ACASI appears to be a useful tool for high risk behaviour assessments in the African context.     

Hepcidin expression and iron transport in alveolar macrophages

Alveolar macrophages express many proteins important in iron homeostasis, including the iron importer divalent metal transport 1 (DMT1) and the iron exporter ferroportin 1 (FPN1) that likely participate in lung defense. We found the iron regulatory hormone hepcidin (HAMP) is also produced by alveolar macrophages. In mouse alveolar macrophages, HAMP mRNA was detected at a low level when not stimulated but at a high level when exposed to lipopolysaccharide (LPS). LPS also affected the mRNA levels of the iron transporters, with DMT1 being upregulated and FPN1 downregulated. However, iron had no effect on HAMP expression but was able to upregulate both DMT1 and FPN1 in alveolar macrophages. IL-1 and IL-6, which are important in HAMP augmentation in hepatocytes, also did not affect HAMP expression in alveolar macrophages. In fact, the LPS-induced alterations in the expression of HAMP as well as DMT1 and FPN1 were preserved in the alveolar macrophages isolated from IL-1 receptor or IL-6-deficient mice. When alveolar macrophages were loaded with transferrin-bound (55)Fe, the subsequent release of (55)Fe was inhibited significantly by LPS. In addition, treatment of these cells with either LPS or HAMP caused the diminishment of the surface FPN1. These findings are consistent with the current model that HAMP production leads to a decreased iron efflux. Our studies suggest that iron mobilization by alveolar macrophages can be affected by iron and LPS via several pathways, including HAMP-mediated degradation of FPN1, and that these cells may use unique regulatory mechanisms to cope with iron imbalance in the lung.     

Fossil avian eggshell preserves ancient DNA

Owing to exceptional biomolecule preservation, fossil avian eggshell has been used extensively in geochronology and palaeodietary studies. Here, we show, to our knowledge, for the first time that fossil eggshell is a previously unrecognized source of ancient DNA (aDNA). We describe the successful isolation and amplification of DNA from fossil eggshell up to 19 ka old. aDNA was successfully characterized from eggshell obtained from New Zealand (extinct moa and ducks), Madagascar (extinct elephant birds) and Australia (emu and owl). Our data demonstrate excellent preservation of the nucleic acids, evidenced by retrieval of both mitochondrial and nuclear DNA from many of the samples. Using confocal microscopy and quantitative PCR, this study critically evaluates approaches to maximize DNA recovery from powdered eggshell. Our quantitative PCR experiments also demonstrate that moa eggshell has approximately 125 times lower bacterial load than bone, making it a highly suitable substrate for high-throughput sequencing approaches. Importantly, the preservation of DNA in Pleistocene eggshell from Australia and Holocene deposits from Madagascar indicates that eggshell is an excellent substrate for the long-term preservation of DNA in warmer climates. The successful recovery of DNA from this substrate has implications in a number of scientific disciplines; most notably archaeology and palaeontology, where genotypes and/or DNA-based species identifications can add significantly to our understanding of diets, environments, past biodiversity and evolutionary processes.     

The effect of inappropriate calibration: three case studies in molecular ecology

Time-scales estimated from sequence data play an important role in molecular ecology. They can be used to draw correlations between evolutionary and palaeoclimatic events, to measure the tempo of speciation, and to study the demographic history of an endangered species. In all of these studies, it is paramount to have accurate estimates of time-scales and substitution rates. Molecular ecological studies typically focus on intraspecific data that have evolved on genealogical scales, but often these studies inappropriately employ deep fossil calibrations or canonical substitution rates (e.g., 1% per million years for birds and mammals) for calibrating estimates of divergence times. These approaches can yield misleading estimates of molecular time-scales, with significant impacts on subsequent evolutionary and ecological inferences. We illustrate this calibration problem using three case studies: avian speciation in the late Pleistocene, the demographic history of bowhead whales, and the Pleistocene biogeography of brown bears. For each data set, we compare the date estimates that are obtained using internal and external calibration points. In all three cases, the conclusions are significantly altered by the application of revised, internally-calibrated substitution rates. Collectively, the results emphasise the importance of judicious selection of calibrations for analyses of recent evolutionary events.     

Temperature-dependent Development of Four Egg Parasitoid Trichogramma Species (Hymenoptera: Trichogrammatidae)

Trichogramma species have been successfully utilized for biocontrol of several lepidopteran pests worldwide. The development, survival and progeny production of two Kenyan species' Trichogramma bournieri Pintureau & Babault and Trichogramma sp. nr. mwanzai Schulten & Feijen (collected from Kenya), Trichogramma evanescens Westwood (Germany) and Trichogramma chilonis Ishii (India) was studied at four constant temperatures (13, 18, 25 and 34°C) with the 011 aim of assessing the relative biotic potential of the two native species for 011 biocontrol of Helicoverpa armigera and Plutella xylostella in Kenya. The study was conducted at the Institute 011 for Biological Control (BBA), Darmstadt, Germany. The Trichogramma species tested showed variations 011 in fertility, developmental time, percent emergence, progeny production and sex ratio 011 at the four temperature regimes. Fertility decreased as temperature increased from 25 to 34°C. 011 T. chilonis and T. evanescens completed development at all temperatures tested, but T. 011 bournieri and T. sp. nr. mwanzai failed to complete development at 13°C. The developmental 011 period for all the species decreased as the temperature increased. The duration of development from 011 oviposition to adult emergence varied from 8 days to 12 weeks shorter at 34°C than at 011 13°C for T. chilonis and T. evanescens . For the various temperatures tested, a linear model was 011 satisfactory for egg to adult development at P = 0.05 for T. chilonis and T. evanescens . The 011 lower temperature thresholds for development and duration in degree-days were 8.83°C and 188 for 011 T. chilonis and 9.23°C and 192 for T. evanescens , respectively. For all temperatures tested, 011 T. sp. nr. mwanzai had the highest preimaginal survivorship. Adult emergence was lower at 13°C and 34°C than at 011 18 and 25°C. The highest fertility (mean ±SE) (50.37 ±2.32 adult 011 female -1 ) and progeny production (44.03 ±2.02 adult female -1 ) was recorded at 25°C for 011 T. evanescens . Sex ratio was biased towards female at all temperatures in T. bournieri and T. chilonis . 011 At all temperatures tested, T . sp. nr. mwanzai produced more males than females. For all species tested, 011 favourable parasitism was between 18 and 25°C. The results from this study will be useful for mass rearing purposes as well as for future field release programmes.     

Characterizing restriction enzyme‐associated loci in historic ragweed (Ambrosia artemisiifolia) voucher specimens using custom‐designed RNA probes

Population genetic studies of nonmodel organisms frequently employ reduced representation library (RRL) methodologies, many of which rely on protocols in which genomic DNA is digested by one or more restriction enzymes. However, because high molecular weight DNA is recommended for these protocols, samples with degraded DNA are generally unsuitable for RRL methods. Given that ancient and historic specimens can provide key temporal perspectives to evolutionary questions, we explored how custom‐designed RNA probes could enrich for RRL loci (Restriction Enzyme‐Associated Loci baits, or REALbaits). Starting with genotyping‐by‐sequencing (GBS) data generated on modern common ragweed (Ambrosia artemisiifolia L.) specimens, we designed 20 000 RNA probes to target well‐characterized genomic loci in herbarium voucher specimens dating from 1835 to 1913. Compared to shotgun sequencing, we observed enrichment of the targeted loci at 19‐ to 151‐fold. Using our GBS capture pipeline on a data set of 38 herbarium samples, we discovered 22 813 SNPs, providing sufficient genomic resolution to distinguish geographic populations. For these samples, we found that dilution of REALbaits to 10% of their original concentration still yielded sufficient data for downstream analyses and that a sequencing depth of ~7m reads was sufficient to characterize most loci without wasting sequencing capacity. In addition, we observed that targeted loci had highly variable rates of success, which we primarily attribute to similarity between loci, a trait that ultimately interferes with unambiguous read mapping. Our findings can help researchers design capture experiments for RRL loci, thereby providing an efficient means to integrate samples with degraded DNA into existing RRL data sets.     

Prognostic value of MIB-1 proliferation index in solitary fibrous tumors of the pleura implemented in a new score – a multicenter study

Background

Although the majority of solitary fibrous tumors of the pleura (SFTP) follow a benign course, 10–25% of patients suffer from recurrence or metastatic disease. Several scoring models have been proposed to predict the outcome. However, none of these included immunohistochemical (IHC) markers as possible prognosticators.

Methods

In this multicenter study, we collected clinical data and formalin-fixed and paraffin-embedded (FFPE) tissue blocks of patients with histologically proven SFTP which had been surgically resected between 2000 und 2015. After systematic and extensive IHC staining on tissue microarrays, the results were analyzed and compared to histomorphological and clinical data for their possible prognostic value.

Results

In total, 78 patients (mean age 61?±?11 years) were included. Of these, 9 patients (11%) had an adverse outcome including SFTP recurrence (n?=?6) or SFTP-related death (n?=?3). Mean overall survival was 172?±?13 months. 1 and 10-year event-free survival rates were 99% and 93%. In the multivariable analysis only MIB-1 proliferation index (Ki-67) ≥10% (HR 12.3, CI 1.1–139.5, p?=?0.043), ≥4 mitoses per 10 high power fields (HR 36.5, CI 1.2–1103.7, p?=?0.039) and tumor size larger than 10 cm (HR 81.8, CI 1.7–4016.8, p?=?0.027) were independently associated with adverse outcome.

Conclusion

A high proliferation rate by MIB-1 IHC was associated with impaired outcome. Upon this, we established a new score using mitosis, necrosis, size of the tumor and MIB-1, which performed better than the traditional scores in our data set. This prognostic score could help to better evaluate outcome of SFTP, but requires external validation.