How many species of Paradoxurus civets are there? New insights from India and Sri Lanka

Using molecular data and morphological features, we investigated the species limits and genetic diversity among populations of the Asian palm civets of the genus Paradoxurus. Our main objectives were to determine the number of species within Paradoxurus hermaphroditus and to test the validity of the newly proposed species within Paradoxurus zeylonensis. Fragments of two mitochondrial (Cytochrome b, Control Region) and one nuclear (intron 7 of the beta fibrinogen) markers were sequenced from 128 individuals of P. hermaphroditus, P. zeylonensis and Paradoxurus jerdoni. DNA sequences were analysed using phylogenetic and haplotype network methods. Our analyses confirmed that P. hermaphroditus comprises three major clades, which should be recognized as separate species: P. hermaphroditus (Indian and Indochinese regions), Paradoxurus musangus (mainland Southeast Asia, Sumatra, Java and other small Indonesian islands) and Paradoxurus philippinensis (Mentawai Islands, Borneo and the Philippines). Furthermore, we have proposed that there are two subspecies within both P. musangus and P. philippinensis, and there might be at least two or three subspecies within P. hermaphroditus. We found a very low genetic diversity and no geographical structure within P. zeylonensis and did not find any support for splitting P. zeylonensis into several species nor subspecies. Finally, we confirmed that P. jerdoni and P. zeylonensis are sister species.     

Sprint,agility, strength and endurance capacity in wheelchair basketball players

The aims of the present study were, firstly, to determine the reliability and reproducibility of an agility T-test and Yo-Yo 10 m recovery test; and secondly, to analyse the physical characteristics measured by sprint, agility, strength and endurance field tests in wheelchair basketball (WB) players. 16 WB players (33.06 ± 7.36 years, 71.89 ± 21.71 kg and sitting body height 86.07 ± 6.82 cm) belonging to the national WB league participated in this study. Wheelchair sprint (5 and 20 m without ball, and 5 and 20 m with ball) agility (T-test and pick-up test) strength (handgrip and maximal pass) and endurance (Yo-Yo 10 m recovery test) were performed. T-test and Yo-Yo 10 m recovery test showed good reproducibility values (intraclass correlation coefficient, ICC = 0.74-0.94). The WB players’ results in 5 and 20 m sprints without a ball were 1.87 ± 0.21 s and 5.70 ± 0.43 s and with a ball 2.10 ± 0.30 s and 6.59 ± 0.61 s, being better than those reported in the literature. Regarding the pick-up test results (16.05 ± 0.52 s) and maximal pass (8.39 ± 1.77 m), players showed worse values than those obtained in elite players. The main contribution of the present study is the characterization of the physical performance profile of WB players using a field test battery. Furthermore, we demonstrated that the agility T-test and the aerobic Yo-Yo 10 m recovery test are reliable; consequently they may be appropriate instruments for measuring physical fitness in WB.     

Ubiquitin fusion expression and tissue-dependent targeting of hG-CSF in transgenic tobacco

Background

The transglutaminase activated factor XIII (FXIIIa) acts to strengthen pathological fibrin clots and to slow their dissolution, in part by crosslinking active α₂-antiplasmin (α₂AP) to fibrin. We previously reported that a yeast-derived recombinant fusion protein comprising α₂AP residues 13-42 linked to human serum albumin (HSA) weakened in vitro clots but failed to become specifically incorporated into in vivo clots. In this study, our aims were to improve both the stability and clot localization of the HSA fusion protein by replacing α₂AP residues 13-42 with shorter sequences recognized more effectively by FXIIIa.

Results

Expression plasmids were prepared encoding recombinant HSA with the following N-terminal 23 residue extensions: H₆NQEQVSPLTLLAG₄Y (designated XL1); H₆DQMMLPWAVTLG₄Y (XL2); H₆WQHKIDLPYNGAG₄Y (XL3); and their 17 residue non-His-tagged equivalents (XL4, XL5, and XL6). The HSA moiety of XL4- to XL6-HSA proteins was C-terminally His-tagged. All chimerae were efficiently secreted from transformed Pichia pastoris yeast except XL3-HSA, and following nickel chelate affinity purification were found to be intact by amino acid sequencing, as was an N-terminally His-tagged version of α₂AP(13-42)-HSA. Of the proteins tested, XL5-HSA was cross-linked to biotin pentylamine (BPA) most rapidly by FXIIIa, and was the most effective competitor of α₂AP crosslinking not only to BPA but also to plasma fibrin clots. In the mouse ferric chloride vena cava thrombosis model, radiolabeled XL5-HSA was retained in the clot to a greater extent than recombinant HSA. In the rabbit jugular vein stasis thrombosis model, XL5-HSA was also retained in the clot, in a urea-insensitive manner indicative of crosslinking to fibrin, to a greater extent than recombinant HSA.

Conclusions

Fusion protein XL5-HSA (DQMMLPWAVTLG₄Y-HSAH₆) was found to be more active as a substrate for FXIIIa-mediated transamidation than seven other candidate fusion proteins in vitro. The improved stability and reactivity of this chimeric protein was further evidenced by its incorporation into in vivo clots formed in thrombosis models in both mice and rabbits.     

Histopathological and parasitological study of the gastrointestinal tract of dogs naturally infected with Leishmania infantum

Background

A nationwide survey on the microbial etiology of cases of subclinical mastitis in dairy cows was carried out on dairy farms in Sweden. The aim was to investigate the microbial panorama and the occurrence of antimicrobial resistance. Moreover, differences between newly infected cows and chronically infected cows were investigated.

Methods

In total, 583 quarter milk samples were collected from 583 dairy cows at 226 dairy farms from February 2008 to February 2009. The quarter milk samples were bacteriological investigated and scored using the California Mastitis Test. Staphylococci were tested for betalactamase production and presence of resistance was evaluated in all specific udder pathogens. Differences between newly infected cows and chronically infected cows were statistically investigated using logistic regression analysis.

Results

The most common isolates of 590 bacteriological diagnoses were Staphylococcus (S) aureus (19%) and coagulase-negative staphylococci (CNS; 16%) followed by Streptococcus (Str) dysgalactiae (9%), Str. uberis (8%), Escherichia (E.) coli (2.9%), and Streptococcus spp. (1.9%). Samples with no growth or contamination constituted 22% and 18% of the diagnoses, respectively. The distribution of the most commonly isolated bacteria considering only bacteriological positive samples were: S. aureus - 31%, CNS - 27%, Str. dysgalactiae - 15%, Str. uberis - 14%, E. coli - 4.8%, and Streptococcus spp. - 3.1%. There was an increased risk of finding S. aureus, Str. uberis or Str. dysgalactiae in milk samples from chronically infected cows compared to findings in milk samples from newly infected cows. Four percent of the S. aureus isolates and 35% of the CNS isolates were resistant to penicillin G. Overall, resistance to other antimicrobials than penicillin G was uncommon.

Conclusions

Staphylococcus aureus and CNS were the most frequently isolated pathogens and resistance to antimicrobials was rare.     

Intensive nitrogen loss over the Omani Shelf due to anammox coupled with dissimilatory nitrite reduction to ammonium

A combination of stable isotopes (¹⁵N) and molecular ecological approaches was used to investigate the vertical distribution and mechanisms of biological N₂ production along a transect from the Omani coast to the central–northeastern (NE) Arabian Sea. The Arabian Sea harbors the thickest oxygen minimum zone (OMZ) in the world''s oceans, and is considered to be a major site of oceanic nitrogen (N) loss. Short (<48 h) anoxic incubations with ¹⁵N-labeled substrates and functional gene expression analyses showed that the anammox process was highly active, whereas denitrification was hardly detectable in the OMZ over the Omani shelf at least at the time of our sampling. Anammox was coupled with dissimilatory nitrite reduction to ammonium (DNRA), resulting in the production of double-¹⁵N-labeled N₂ from ¹⁵NO₂⁻, a signal often taken as the lone evidence for denitrification in the past. Although the central–NE Arabian Sea has conventionally been regarded as the primary N-loss region, low potential N-loss rates at sporadic depths were detected at best. N-loss activities in this region likely experience high spatiotemporal variabilities as linked to the availability of organic matter. Our finding of greater N-loss associated with the more productive Omani upwelling region is consistent with results from other major OMZs. The close reliance of anammox on DNRA also highlights the need to take into account the effects of coupling N-transformations on oceanic N-loss and subsequent N-balance estimates.     

Anaerobic ammonia oxidation in a fertilized paddy soil

Evidence for anaerobic ammonium oxidation in a paddy field was obtained in Southern China using an isotope-pairing technique, quantitative PCR assays and 16S rRNA gene clone libraries, along with nutrient profiles of soil cores. A paddy field with a high load of slurry manure as fertilizer was selected for this study and was shown to contain a high amount of ammonium (6.2–178.8 mg kg⁻¹). The anaerobic oxidation of ammonium (anammox) rates in this paddy soil ranged between 0.5 and 2.9 nmolN per gram of soil per hour in different depths of the soil core, and the specific cellular anammox activity observed in batch tests ranged from 2.9 to 21 fmol per cell per day. Anammox contributed 4–37% to soil N2 production, the remainder being due to denitrification. The 16S rRNA gene sequences of surface soil were closely related to the anammox bacteria ‘Kuenenia'', ‘Anammoxoglobus'' and ‘Jettenia''. Most of the anammox 16S rRNA genes retrieved from the deeper soil were affiliated to ‘Brocadia''. The retrieval of mainly bacterial amoA sequences in the upper part of the paddy soil indicated that nitrifying bacteria may be the major source of nitrite for anammox bacteria in the cultivated horizon. In the deeper oxygen-limited parts, only archaeal amoA sequences were found, indicating that archaea may produce nitrite in this part of the soil. It is estimated that a total loss of 76 g N m⁻² per year is linked to anammox in the paddy field.     

International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients

Background

A century after its discovery, Chagas disease still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The purpose of this study was to evaluate the performance of PCR methods in detection of Trypanosoma cruzi DNA by an external quality evaluation.

Methodology/Findings

An international collaborative study was launched by expert PCR laboratories from 16 countries. Currently used strategies were challenged against serial dilutions of purified DNA from stocks representing T. cruzi discrete typing units (DTU) I, IV and VI (set A), human blood spiked with parasite cells (set B) and Guanidine Hidrochloride-EDTA blood samples from 32 seropositive and 10 seronegative patients from Southern Cone countries (set C). Forty eight PCR tests were reported for set A and 44 for sets B and C; 28 targeted minicircle DNA (kDNA), 13 satellite DNA (Sat-DNA) and the remainder low copy number sequences. In set A, commercial master mixes and Sat-DNA Real Time PCR showed better specificity, but kDNA-PCR was more sensitive to detect DTU I DNA. In set B, commercial DNA extraction kits presented better specificity than solvent extraction protocols. Sat-DNA PCR tests had higher specificity, with sensitivities of 0.05–0.5 parasites/mL whereas specific kDNA tests detected 5.10⁻³ par/mL. Sixteen specific and coherent methods had a Good Performance in both sets A and B (10 fg/µl of DNA from all stocks, 5 par/mL spiked blood). The median values of sensitivities, specificities and accuracies obtained in testing the Set C samples with the 16 tests determined to be good performing by analyzing Sets A and B samples varied considerably. Out of them, four methods depicted the best performing parameters in all three sets of samples, detecting at least 10 fg/µl for each DNA stock, 0.5 par/mL and a sensitivity between 83.3–94.4%, specificity of 85–95%, accuracy of 86.8–89.5% and kappa index of 0.7–0.8 compared to consensus PCR reports of the 16 good performing tests and 63–69%, 100%, 71.4–76.2% and 0.4–0.5, respectively compared to serodiagnosis. Method LbD2 used solvent extraction followed by Sybr-Green based Real time PCR targeted to Sat-DNA; method LbD3 used solvent DNA extraction followed by conventional PCR targeted to Sat-DNA. The third method (LbF1) used glass fiber column based DNA extraction followed by TaqMan Real Time PCR targeted to Sat-DNA (cruzi 1/cruzi 2 and cruzi 3 TaqMan probe) and the fourth method (LbQ) used solvent DNA extraction followed by conventional hot-start PCR targeted to kDNA (primer pairs 121/122). These four methods were further evaluated at the coordinating laboratory in a subset of human blood samples, confirming the performance obtained by the participating laboratories.

Conclusion/Significance

This study represents a first crucial step towards international validation of PCR procedures for detection of T. cruzi in human blood samples.     

Biomechanical Energy‐Driven Hybridized Generator as a Universal Portable Power Source for Smart/Wearable Electronics

The fast growth of smart electronics requires novel solutions to power them sustainably. Portable power sources capable of harvesting biomechanical energy are a promising modern approach to reduce battery dependency. Herein, a novel elastic impact‐based nonresonant hybridized generator (EINR‐HG) is reported to effectively harvest biomechanical energy from diverse human activities outdoors. Through the rational integration of a nonlinear electromagnetic generator with two contact‐mode triboelectric nanogenerators, the proposed EINR‐HG generates hybrid electrical output simultaneously under the same mechanical excitations. By introducing a flux‐concentrator with a nanowire‐nanofiber surface modification, significant improvement in the energy harvesting efficiency of the EINR‐HG is achieved. After optimizing using simulations and vibration tests, the as‐fabricated EINR‐HG delivers an outstanding normalized power density of 3.13 mW cm^?3 g^?2 across a matching resistance of 1.5 kΩ at 6 Hz under 1 g acceleration. Under human motion testing, the EINR‐HG generates an optimal output power of 131.4 mW with horizontal handshaking. With a customized power management circuit, the EINR‐HG serves as a universal power source that successfully drives commercial smart electronics, including smart bands and smartphones. This work shows the massive potential of biomechanical energy‐driven hybridized generators for powering personal electronics and portable healthcare monitoring devices.     

New insights on the geographical origins of the Caribbean raccoons

The introduction of species outside their natural range is one of the major threats to biodiversity and has often been identified as a menace to agricultural production and human health. The raccoon is recognized as a globally invasive species. However, several populations in the Caribbean were long considered native and endemic species. Although previous genetic studies have shown that raccoons from the islands of the West Indies belong to the northern raccoon Procyon lotor, the history and origin of these introductions remain poorly known. In this study, we investigated the geographical origin of Caribbean raccoon populations using newly available molecular genetic data. We used haplotype network analyses of two mitochondrial markers, Cytochrome b and Control Region, with new sequences and those from GenBank. We also specifically investigated the origin of the endangered endemic Cozumel raccoon, Procyon pygmaeus, by re-analyzing data. Our results confirmed that all Caribbean raccoon populations belong to the northern raccoon. Bahamian populations originated from two different sources in Florida, and the Lesser Antilles raccoons seem to originate from northern regions of the native range. In addition, our results question the taxonomic status of the Cozumel raccoon, as currently available genetic data support a conspecific status with the northern raccoon. These results have important implications in the context of conservation and ecosystem management. Identifying origins of introduced populations and understanding the history of their introductions will facilitate studies on the impact of the raccoon on insular ecosystems.