Morphological and genetic discordance in two species of Bornean Leptobrachium (Amphibia, Anura, Megophryidae)

Recent phylogenetic studies of Southeast Asian megophryid Leptobrachium, while clarifying (1) distinct specific status of three Philippine populations and (2) high genetic diversities within Bornean Leptobrachium montanum, posed two questions, (1) relationships and divergence histories of two Philippine species and Bornean Leptobrachium gunungense, and (2) possible discordance between phylogenetically and morphologically defined lineages. In order to solve these questions, and especially reviewing current taxonomy of Bornean species, we estimated the phylogenetic relationships of endemic Bornean species together with their putative relatives from Philippines and Sumatra, using 2451 bp sequences of the 12S rRNA, tRNA(val), and 16S rRNA of mitochondrial DNA genes. With respect to Leptobrachium hasseltii and Leptobrachium chapaense, lineages from Borneo, Philippines, and Sumatra formed a monophyletic group with Leptobrachium lumadorum from Mindanao as the basal clade, while two other Philippine species from Palawan and Mindoro formed a clade and nested in Bornean lineages. Sister species relationship of the two Philippine species and L. gunungense is not supported, rejecting the hypothesis of Philippine origin of L. gunungense. Phylogeny does not conform to morphological variation, and the topotypic L. montanum and one lineage of Leptobrachium abbotti are genetically very close despite their clear difference in ventral color pattern. Furthermore, each of these species forms a paraphyletic group and contains several lineages, each of which is a candidate of good species. These results clearly indicate that current taxonomy of Bornean species based on several morphological characteristics requires complete revision. Detailed studies on adult and larval morphology, as well as acoustic characteristics, are necessary to evaluate the taxonomic status of all lineages recovered.     

IL-1β stimulates COX-2 dependent PGE₂ synthesis and CGRP release in rat trigeminal ganglia cells

Objective

Pro-inflammatory cytokines like Interleukin-1 beta (IL-1β) have been implicated in the pathophysiology of migraine and inflammatory pain. The trigeminal ganglion and calcitonin gene-related peptide (CGRP) are crucial components in the pathophysiology of primary headaches. 5-HT1B/D receptor agonists, which reduce CGRP release, and cyclooxygenase (COX) inhibitors can abort trigeminally mediated pain. However, the cellular source of COX and the interplay between COX and CGRP within the trigeminal ganglion have not been clearly identified.

Methods and Results

1. We used primary cultured rat trigeminal ganglia cells to assess whether IL-1β can induce the expression of COX-2 and which cells express COX-2. Stimulation with IL-1β caused a dose and time dependent induction of COX-2 but not COX-1 mRNA. Immunohistochemistry revealed expression of COX-2 protein in neuronal and glial cells. 2. Functional significance was demonstrated by prostaglandin E2 (PGE₂) release 4 hours after stimulation with IL-1β, which could be aborted by a selective COX-2 (parecoxib) and a non-selective COX-inhibitor (indomethacin). 3. Induction of CGRP release, indicating functional neuronal activation, was seen 1 hour after PGE₂ and 24 hours after IL-1β stimulation. Immunohistochemistry showed trigeminal neurons as the source of CGRP. IL-1β induced CGRP release was blocked by parecoxib and indomethacin, but the 5-HT1B/D receptor agonist sumatriptan had no effect.

Conclusion

We identified a COX-2 dependent pathway of cytokine induced CGRP release in trigeminal ganglia neurons that is not affected by 5-HT1B/D receptor activation. Activation of neuronal and glial cells in the trigeminal ganglion by IL-β leads to an elevated expression of COX-2 in these cells. Newly synthesized PGE₂ (by COX-2) in turn activates trigeminal neurons to release CGRP. These findings support a glia-neuron interaction in the trigeminal ganglion and demonstrate a sequential link between COX-2 and CGRP. The results could help to explain the mechanism of action of COX-2 inhibitors in migraine.     

Determining Mycobacterium tuberculosis Infection among BCG-Immunised Ugandan Children by T-SPOT.TB and Tuberculin Skin Testing

Background

Children with latent tuberculosis infection (LTBI) represent a huge reservoir for future disease. We wished to determine Mycobacterium tuberculosis (M.tb) infection prevalence among BCG-immunised five-year-old children in Entebbe, Uganda, but there are limited data on the performance of immunoassays for diagnosis of tuberculosis infection in children in endemic settings. We therefore evaluated agreement between a commercial interferon gamma release assay (T-SPOT.TB) and the tuberculin skin test (TST; 2 units RT-23 tuberculin; positive defined as diameter ≥10 mm), along with the reproducibility of T-SPOT.TB on short-term follow-up, in this population.

Methodology/Principal Findings

We recruited 907 children of which 56 were household contacts of TB patients. They were tested with T-SPOT.TB at age five years and then re-examined with T-SPOT.TB (n = 405) and TST (n = 319) approximately three weeks later. The principal outcome measures were T-SPOT.TB and TST positivity. At five years, 88 (9.7%) children tested positive by T-SPOT.TB. More than half of those that were T-SPOT.TB positive at five years were negative at follow-up, whereas 96% of baseline negatives were consistently negative. We observed somewhat better agreement between initial and follow-up T-SPOT.TB results among household TB contacts (κ = 0.77) than among non-contacts (κ = 0.39). Agreement between T-SPOT.TB and TST was weak (κ = 0.28 and κ = 0.40 for T-SPOT.TB at 5 years and follow-up, respectively). Of 28 children who were positive on both T-SPOT.TB tests, 14 (50%) had a negative TST. Analysis of spot counts showed high levels of instability in responses between baseline and follow-up, indicating variability in circulating numbers of T cells specific for certain M.tb antigens.

Conclusions/Significance

We found that T-SPOT.TB positives are unstable over a three-week follow-up interval, and that TST compares poorly with T-SPOT.TB, making the categorisation of children as TB-infected or TB-uninfected difficult. Existing tools for the diagnosis of TB infection are unsatisfactory in determining infection among children in this setting.     

Reproduction and feeding habits of the highly seasonal Brachyhypopomus bombilla (Gymnotiformes: Hypopomidae) from southern Brazil with evidence for a dormancy period

The reproductive biology and feeding habits of the hypopomid Brachyhypopomus bombilla were studied. The species has seasonal behavior, with breeding and feeding activity occurring mostly during the Southern Hemisphere spring and summer. We defined fractional spawning for the species and a relative fecundity similar to those previously reported for other gymnotiform species. According to the analysis of the food items, B. bombilla was included in the trophic category invertivorous, feeding mainly on autochthonous insects. The reproductive periods of males and females as well as female feeding activity were significantly related to the photoperiod. During a period of 5?months, when the lowest water temperatures and shortest photoperiod were recorded, all the specimens of B. bombilla were found buried in the muddy bottom of the creek, with lower electric organ discharge activity, empty stomachs, and gonads in early stages of the maturation process. This period, herein defined as a dormancy period, is reported here for the first time for a species of the Gymnotiformes order.     

Sublingual immunization with an engineered Bacillus subtilis strain expressing tetanus toxin fragment C induces systemic and mucosal immune responses in piglets

Sublingual (SL) and intranasal (IN) administration of a Bacillus subtilis-based tetanus vaccine was tested in piglets, which more closely mimic the human immune system than mice. Piglets were immunized by the SL, IN or oral routes with vaccine expressing tetanus toxin fragment C, or commercial tetanus vaccine given by intramuscular injection as a control. Tetanus toxoid specific ELISA and passive neutralization tests were used to measure IgG and IgA levels in serum and mucosal secretions, and assess protective serum antibodies, respectively. The nature of the immune response was explored by MHC Class II, TGF-β1 expression, and ELISA assays for multiple cytokines. SL or IN immunization of piglets induced neutralizing tetanus toxoid specific serum antibody and local salivary and vaginal IgA responses. Standard tetanus vaccine resulted in systemic antibodies, whereas oral administration of the Bacillus-based vaccine was ineffective. Further analyses indicated a balanced Th1/Th2 response to SL or IN immunization. CONCLUSION: This study demonstrates for the first time that SL or IN administration is effective for inducing both systemic and mucosal responses in a piglet model, indicating that SL or IN delivery of a B. subtilis-based tetanus vaccine can be a simple, non-invasive, low cost strategy to induce immunity to tetanus.     

Impact of sea-level and climatic changes on the Amazon coastal wetlands during the late Holocene

Wetland dynamics in northern Brazil during the Holocene were studied by pollen analysis and AMS radiocarbon dating of three cores. Near the Amazon mouth region, covered mainly by primary Amazon coastal forest and herbaceous vegetation, the pollen record indicates the dominance of mangroves between 4800 and 1100 cal yr b.p. A contraction of the mangrove area and an expansion of herbaceous and fern vegetation occurred between 1100 and 750 cal yr b.p. The period between 750 and 200 cal yr b.p. is characterized by an expansion of mangrove and a decrease in herbaceous and fern vegetation. This trend continued until the present. On Atalaia Island, the sediment core indicates a period with poor pollen preservation between 830 and 630 cal yr b.p. Between 630 and 330 cal yr b.p., mangroves expanded. Later, up to 45 cal yr b.p., the mangrove area decreased and the herbaceous vegetation expanded. During the last hundred years, the relative sea-level rise most probably favored the mangrove expansion as far as the topographically highest sector on this island, while the herbaceous vegetation decreased. The pollen data from água Preta Lake indicate dry conditions, as reflected by the poor pollen preservation between 390 and 240 cal yr b.p. Between 240 and 60 cal yr b.p., restinga and Amazon coastal forest with palms dominated this region. For the last 120 years, the record indicates an expansion of the mangrove area. However, recent confinement of mangrove development to the topographically highest area, and the loss of mangrove areas on the lowest surfaces have led to a net loss of mangrove coverage during the last decades.     

Incidence and clinical characteristics of group A rotavirus infections among children admitted to hospital in Kilifi, Kenya

Background

Rotavirus, predominantly of group A, is a major cause of severe diarrhoea worldwide, with the greatest burden falling on young children living in less-developed countries. Vaccines directed against this virus have shown promise in recent trials, and are undergoing effectiveness evaluation in sub-Saharan Africa. In this region limited childhood data are available on the incidence and clinical characteristics of severe group A rotavirus disease. Advocacy for vaccine intervention and interpretation of effectiveness following implementation will benefit from accurate base-line estimates of the incidence and severity of rotavirus paediatric admissions in relevant populations. The study objective was to accurately define the incidence and severity of group A rotavirus disease in a resource-poor setting necessary to make informed decisions on the need for vaccine prevention.

Methods and Findings

Between 2002 and 2004 we conducted prospective surveillance for group A rotavirus infection at Kilifi District Hospital in coastal Kenya. Children < 13 y of age were eligible as “cases” if admitted with diarrhoea, and “controls” if admitted without diarrhoea. We calculated the incidence of hospital admission with group A rotavirus using data from a demographic surveillance study of 220,000 people in Kilifi District. Of 15,347 childhood admissions 3,296 (22%) had diarrhoea, 2,039 were tested for group A rotavirus antigen and, of these, 588 (29%) were positive. 372 (63%) rotavirus-positive cases were infants. Of 620 controls 19 (3.1%, 95% confidence interval [CI] 1.9–4.7) were rotavirus positive. The annual incidence (per 100,000 children) of rotavirus-positive admissions was 1,431 (95% CI 1,275–1,600) in infants and 478 (437–521) in under-5-y-olds, and highest proximal to the hospital. Compared to children with rotavirus-negative diarrhoea, rotavirus-positive cases were less likely to have coexisting illnesses and more likely to have acidosis (46% versus 17%) and severe electrolyte imbalance except hyponatraemia. In-hospital case fatality was 2% among rotavirus-positive and 9% among rotavirus-negative children.

Conclusions

In Kilifi > 2% of children are admitted to hospital with group A rotavirus diarrhoea in the first 5 y of life. This translates into over 28,000 vaccine-preventable hospitalisations per year across Kenya, and is likely to be a considerable underestimate. Group A rotavirus diarrhoea is associated with acute life-threatening metabolic derangement in otherwise healthy children. Although mortality is low in this clinical research setting this may not be generally true in African hospitals lacking rapid and appropriate management.     

Synthesis and characterization of cross-linked malonylchitosan microspheres for controlled release of acyclovir

The purpose of the present study was to obtain a polymeric system for delayed release of the drug acyclovir (ACV), which can be used for treatment of Herpes simplex and Varicella Zoster. The gelled chitosan (GCT) microspheres were obtained by coacervation-phase separation. They were treated with malonic acid to obtain malonylchitosan (MLCT) microspheres, which were characterized by, Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance spectroscopy (¹³C NMR), elemental analysis (CHN), thermogravimetric analysis (TG/DTG) and scanning electron microscopy (SEM). The drug was encapsulated in MLCT microspheres by a contact adsorption technique, and the final formulation (MLCT-ACV), was analyzed for loading efficiency, degree of swelling and in vitro release profiles. The results obtained support the N-substitution of malonyl groups in the MLCT microspheres. The loading efficiency increased with impregnation time and a major amount of drug was encapsulated after 24 h. The swelling rate was higher in acid pH. The median release time was 5.5 h in pH 1.2 and 6.8. The mechanism involved in release was non-Fickian (0.43 < n < 0.85, n = 0.8474) and Super Case II kinetics (n > 1, n = 1.0491) at pH 1.2 and 6.8, respectively.     

Ribosomal position and contacts of mRNA in eukaryotic translation initiation complexes

The position of mRNA on 40S ribosomal subunits in eukaryotic initiation complexes was determined by UV crosslinking using mRNAs containing uniquely positioned 4-thiouridines. Crosslinking of mRNA positions (+)11 to ribosomal protein (rp) rpS2(S5p) and rpS3(S3p), and (+)9-(+)11 and (+)8-(+)9 to h18 and h34 of 18S rRNA, respectively, indicated that mRNA enters the mRNA-binding channel through the same layers of rRNA and proteins as in prokaryotes. Upstream of the P-site, the proximity of positions (-)3/(-)4 to rpS5(S7p) and h23b, (-)6/(-)7 to rpS14(S11p), and (-)8-(-)11 to the 3'-terminus of 18S rRNA (mRNA/rRNA elements forming the bacterial Shine-Dalgarno duplex) also resembles elements of the bacterial mRNA path. In addition to these striking parallels, differences between mRNA paths included the proximity in eukaryotic initiation complexes of positions (+)7/(+)8 to the central region of h28, (+)4/(+)5 to rpS15(S19p), and (-)6 and (-)7/(-)10 to eukaryote-specific rpS26 and rpS28, respectively. Moreover, we previously determined that eukaryotic initiation factor2alpha (eIF2alpha) contacts position (-)3, and now report that eIF3 interacts with positions (-)8-(-)17, forming an extension of the mRNA-binding channel that likely contributes to unique aspects of eukaryotic initiation.