Non-invasive sex identification of the white-bellied sea eagle (Haliaeetus leucogaster) through genetic analysis of feathers

The white-bellied sea eagle, Haliaeetus leucogaster, displays reversed sexual size dimorphism and is monomorphic for adult plumage coloration. Early attempts to identify sex in sexually monomorphic birds were based on morphological or chromosomal characters, but since avian W-specific DNA sequences were identified, PCR amplification has become commonly used for molecular sexing. We used a PCR test employing primers that amplify two homologous fragments of both the CHD-W gene, unique to females, and the CHD-Z gene, occurring in both sexes. This test was applied to five individuals of H. leucogaster from the Malacca Zoo and to male and female domestic chickens, Gallus domesticus, for comparison. All individuals were sexed successfully with high reproducibility. We conclude that this PCR-based test with feathers as the DNA source is a reliable sexing method for H. leucogaster. This sexing technique is objective and non-invasive and could be used to test sex ratio theories, as well as to help improve conservation and management actions for captive breeding program of this species in Malaysia.     

Complete killing of Caenorhabditis elegans by Burkholderia pseudomallei is dependent on prolonged direct association with the viable pathogen

Background

Burkholderia pseudomallei is the causative agent of melioidosis, a disease of significant morbidity and mortality in both human and animals in endemic areas. Much remains to be known about the contributions of genotypic variations within the bacteria and the host, and environmental factors that lead to the manifestation of the clinical symptoms of melioidosis.

Methodology/Principal Findings

In this study, we showed that different isolates of B. pseudomallei have divergent ability to kill the soil nematode Caenorhabditis elegans. The rate of nematode killing was also dependent on growth media: B. pseudomallei grown on peptone-glucose media killed C. elegans more rapidly than bacteria grown on the nematode growth media. Filter and bacteria cell-free culture filtrate assays demonstrated that the extent of killing observed is significantly less than that observed in the direct killing assay. Additionally, we showed that B. pseudomallei does not persistently accumulate within the C. elegans gut as brief exposure to B. pseudomallei is not sufficient for C. elegans infection.

Conclusions/Significance

A combination of genetic and environmental factors affects virulence. In addition, we have also demonstrated that a Burkholderia-specific mechanism mediating the pathogenic effect in C. elegans requires proliferating B. pseudomallei to continuously produce toxins to mediate complete killing.     

A secretory PLA2 associated with tobacco hornworm hemocyte membrane preparations acts in cellular immune reactions

We report on a secretory phospholipase A2 (sPLA2) associated with membrane-enriched fractions prepared from hemocytes of the tobacco hornworms, Manduca sexta. Virtually no PLA2 activity was detected in serum of immunologically naive or bacterially challenged hornworms. PLA2 activity was detected in cytosolic and membrane-enriched fractions prepared from hemocytes. PLA2 activity in the cytosolic fraction (1.2 pmol/mg/h) was approximately 4-fold greater than in the membrane-enriched fraction. The cytosol-associated PLA2 activity was strongly inhibited in reactions conducted in the presence of the specific cytosolic PLA2 inhibitor methylarachidonyl fluorophosphate (MAFP) but not in the presence of the sPLA2 inhibitor p-bromophenacyl bromide (BPB). Conversely, the membrane-associated PLA2 activity was inhibited in reactions conducted in the presence of BPB but not in the presence of MAFP. While the cytosol-associated PLA2 was independent of calcium, the membrane-associated sPLA2 required calcium for full catalytic activity. Hornworms treated with either BPB, MAFP or the glucocorticosteroid dexamethasone were severely impaired (by 50 to 80% relative to controls) in their ability to form nodules in reaction to bacterial challenge. However, the immune-impairing influence of the inhibitors was reversed by treating larvae with arachidonic acid, a precursor for eicosanoid biosynthesis. We infer that the biological significance of the sPLA2 (as well as the previously characterized cytosolic PLA2) relates to hydrolysis of polyunsaturated fatty acids from cellular phospholipids. Moreover, this enzyme may be the target of immunity-impairing factors from the bacterium Xenorhabdus nematophila. The fatty acids serve as precursors for the generation of eicosanoids responsible for mediating and coordinating cellular immune reactions to infection.     

Diverse signaling pathways through the SDF-1/CXCR4 chemokine axis in prostate cancer cell lines leads to altered patterns of cytokine secretion and angiogenesis

The establishment of metastatic bone lesions in prostate cancer (CaP) is a process partially dependent on angiogenesis. Previously we demonstrated that the stromal-derived factor-1 (SDF-1 or CXCL12)/CXCR4 chemokine axis is critical for CaP cell metastasis. In this investigation, cell lines were established in which CXCR4 expression was knocked down using siRNA technology. When CaP cells were co-transplanted with human vascular endothelial cells into SCID mice, significantly fewer human blood vessels were observed paralleling the reductions in CXCR4 levels. Likewise, the invasive behaviors of the CaP cells were inhibited in vitro. From these functional observations we explored angiogenic and signaling mechanisms generated following SDF-1 binding to CXCR4. Differential activation of the MEK/ERK and PI3K/AKT pathways that result in differential secretion IL-6, IL-8, TIMP-2 and VEGF were seen contingent on the cell type examined; VEGF and TIMP-2 expression in PC3 cells are dependent on AKT activation and ERK activation in LNCaP and LNCaP C4-2B cells leads to IL-6 or IL-8 secretion. At the same time, expression of angiostatin levels were inversely related to CXCR4 levels, and inhibited by SDF-1 stimulation. These data link the SDF-1/CXCR4 pathway to changes in angiogenic cytokines by different signaling mechanisms and, suggest that the delicate equilibrium between proangiogenic and antiangiogenic factors may be achieved by different signal transduction pathways to regulate the angiogenic phenotype of prostate cancers. Taken together, our results provide new information regarding expression of functional CXCR4 receptor-an essential role and potential mechanism of angiogenesis upon SDF-1 stimulation.     

Lateral angle: a method for sexing using the petrous bone

We report on the results of applying the so-called lateral angle method for sex determination on skeletal remains. The lateral angle denotes the angle of the internal auditory canal in relation to the medial surface of the petrous part of the temporal bone. The method involves making a small cast of the proximal part of the internal acoustic canal and determining the angle at which the canal opens up to the surface of the petrous bone. The method has the great advantage of utilizing one of the sturdiest bone elements of the human skeleton, and may thus be especially suited for analyses of very fragmented skeletal remains or cremated bones, where the petrous bone may still be readily recognizable. The method was tested using a forensic sample of 113 petrous bones with known sex. Intra- and interobserver testing was also performed. We found a statistically significant difference in angle size between males and females (mean angle size of males, 39.3 degrees ; mean angle size of females, 48.2 degrees ; P < 0.001). There was no bilateral difference in angle size. In blind trials, 83.2% of petrous bones were assigned to the correct sex. We also tested the lateral angle method against an archaeological skeletal sample. True sex was not known for this sample; instead, sexing had been carried out by assessing pelvic and cranial morphology in independent trials. We found a higher concordance between the lateral angle and "pelvic" sex than for lateral angle and "cranial" sex. Finally, we note that subadult sexing may also be possible with this method.     

A study on medicinal plants from Malaysia focused on Acalypha siamensis Oliv. ex Gage. isolation and structure of a new tetraterpene, acalyphaser A

As a part of our chemical studies on Malaysian medicinal plants, five Malaysian plant species were evaluated by cytotoxicity assays using P388 murine leukemia cells. Since Acalypha siamensis exhibited the strongest growth inhibition, its constituents were studied as the object of search for bioactive materials. A novel tetraterpene, acalyphaser A (1), was isolated in the course of the purification. Its structure was elucidated on the basis of 1D- and 2D-NMR techniques, and mass spectrometry.     

Root colonisation of Pseudomonas aeruginosa strain UPMP3 and induction of defence‐related genes in oil palm (Elaeis guineensis)

Pseudomonas aeruginosa strain UPMP3 labelled with β‐glucuronidase (gusA) and green fluorescent protein (gfp) by electrotransformation yielded ca 1 × 10⁷ transformants µg^?1 DNA. The data obtained from the dilution plate count showed that over 28 days both epiphytic and endophytic populations of P. aeruginosa strain UPMP3 increased from 5.76 log₁₀ [colony forming unit (CFU) + 1] g^?1 fresh weight (FW) to 8.19 log₁₀ (CFU + 1) g^?1 FW and 4.10 log₁₀ (CFU + 1) g^?1 FW to 6.23 (CFU + 1) g^?1 FW, respectively. Confocal laser scanning microscopic analysis of oil palm roots treated with gusA:gfp‐tagged P. aeruginosa strain UPMP3 showed intense root colonisation over the sampling period. The root surface colonisation by P. aeruginosa strain UPMP3 was followed by a second stage, characterised by cortical infection, and a third stage, which involves xylem ingression. The colonisation of oil palm roots by the gusA:gfp‐tagged strain was concentrated on root areas potentially rich in nutrients such as the elongation zones, ridges between epidermal cells and points of secondary adventitious root emergence. Different expression levels of defence‐related genes, namely, chitinase and β‐1,3‐glucanase in the strain UPMP3–host interaction recorded over 28 days, suggested the potential role of P. aeruginosa strain UPMP3 in triggering the defence mechanism in oil palm. This is the first report on root colonisation and upregulation of defence‐related genes on oil palm roots by P. aeruginosa strain UPMP3 and shows the potential of this strain to be used as a biocontrol agent in oil palm.     

Anti-amoebic properties of a Malaysian marine sponge <Emphasis Type="Italic">Aaptos</Emphasis> sp<Emphasis Type="Italic">.</Emphasis> on <Emphasis Type="Italic">Acanthamoeba castellanii</Emphasis>

Crude methanol extracts of a marine sponge, Aaptos aaptos, collected from three different localities namely Kapas, Perhentian and Redang Islands, Terengganu, Malaysia, were tested in vitro on a pathogenic Acanthamoeba castellanii (IMR isolate) to examine their anti-amoebic potential. The examination of anti-Acanthamoebic activity of the extracts was conducted in 24 well plates for 72 h at 30 °C. All extracts possessed anti-amoebic activity with their IC₅₀ values ranging from 0.615 to 0.876 mg/mL. The effect of the methanol extracts on the surface morphology of A. castellanii was analysed under scanning electron microscopy. The ability of the extracts to disrupt the amoeba cell membrane was indicated by extensive cell’s blebbing, changes in the surface morphology, reduced in cell size and with cystic appearance of extract-treated Acanthamoeba. Number of acanthapodia and food cup was also reduced in this Acanthamoeba. Morphological criteria of apoptosis in Acanthamoeba following treatment with the sponge’s extracts was determined by acridine orange-propidium iodide staining and observed by fluorescence microscopy. By this technique, apoptotic and necrotic cells can be visualized and quantified. The genotoxic potential of the methanol extracts was performed by the alkaline comet assay. All methanol extracts used were significantly induced DNA damage compared to untreated Acanthamoeba by having high percentage of scores 1, 2, and 3 of the DNA damage. Results from cytotoxicity and genotoxicity studies carried out in the present study suggest that all methanol extracts of A. aaptos have anti-amoebic properties against A. castellanii.     

Long lasting modulation of cortical oscillations after continuous theta burst transcranial magnetic stimulation

Transcranial magnetic theta burst stimulation (TBS) differs from other high-frequency rTMS protocols because it induces plastic changes up to an hour despite lower stimulus intensity and shorter duration of stimulation. However, the effects of TBS on neuronal oscillations remain unclear. In this study, we used electroencephalography (EEG) to investigate changes of neuronal oscillations after continuous TBS (cTBS), the protocol that emulates long-term depression (LTD) form of synaptic plasticity. We randomly divided 26 healthy humans into two groups receiving either Active or Sham cTBS as control over the left primary motor cortex (M1). Post-cTBS aftereffects were assessed with behavioural measurements at rest using motor evoked potentials (MEPs) and at active state during the execution of a choice reaction time (RT) task in combination with continuous electrophysiological recordings. The cTBS-induced EEG oscillations were assessed using event-related power (ERPow), which reflected regional oscillatory activity of neural assemblies of θ (4-7.5 Hz), low α (8-9.5 Hz), μ (10-12.5 Hz), low β (13-19.5 Hz), and high β (20-30 Hz) brain rhythms. Results revealed 20-min suppression of MEPs and at least 30-min increase of ERPow modulation, suggesting that besides MEPs, EEG has the potential to provide an accurate cortical readout to assess cortical excitability and to investigate the interference of cortical oscillations in the human brain post-cTBS. We also observed a predominant modulation of β frequency band, supporting the hypothesis that cTBS acts more on cortical level. Theta oscillations were also modulated during rest implying the involvement of independent cortical theta generators over the motor network post cTBS. This work provided more insights into the underlying mechanisms of cTBS, providing a possible link between synchronised neural oscillations and LTD in humans.     

DNA Barcoding Reveals Cryptic Diversity within Commercially Exploited Indo-Malay Carangidae (Teleosteii: Perciformes)

Background

DNA barcodes, typically focusing on the cytochrome oxidase I gene (COI) in many animals, have been used widely as a species-identification tool. The ability of DNA barcoding to distinguish species from a range of taxa and to reveal cryptic species has been well documented. Despite the wealth of DNA barcode data for fish from many temperate regions, there are relatively few available from the Southeast Asian region. Here, we target the marine fish Family Carangidae, one of the most commercially-important families from the Indo-Malay Archipelago (IMA), to produce an initial reference DNA barcode library.

Methodology/Principal Findings

Here, a 652 bp region of COI was sequenced for 723 individuals from 36 putative species of Family Carangidae distributed within IMA waters. Within the newly-generated dataset, three described species exhibited conspecific divergences up to ten times greater (4.32–4.82%) than mean estimates (0.24–0.39%), indicating a discrepancy with assigned morphological taxonomic identification, and the existence of cryptic species. Variability of the mitochondrial DNA COI region was compared within and among species to evaluate the COI region''s suitability for species identification. The trend in range of mean K2P distances observed was generally in accordance with expectations based on taxonomic hierarchy: 0% to 4.82% between individuals within species, 0% to 16.4% between species within genera, and 8.64% to 25.39% between genera within families. The average Kimura 2-parameter (K2P) distance between individuals, between species within genera, and between genera within family were 0.37%, 10.53% and 16.56%, respectively. All described species formed monophyletic clusters in the Neighbour-joining phylogenetic tree, although three species representing complexes of six potential cryptic species were detected in Indo-Malay Carangidae; Atule mate, Selar crumenophthalmus and Seriolina nigrofasciata.

Conclusion/Significance

This study confirms that COI is an effective tool for species identification of Carangidae from the IMA. There were moderate levels of cryptic diversity among putative species within the central IMA. However, to explain the hypothesis of species richness in the IMA, it is necessary to sample the whole family across their broad geographic range. Such insights are helpful not only to document mechanisms driving diversification and recruitment in Carangidae, but also to provide a scientific framework for management strategies and conservation of commercially-important fisheries resources.