Dietary protein requirement for captive juvenile green turtles (Chelonia mydas)

Head-starting programs are extremely important for restoring the population of sea turtles in wild whereas husbandry conditions and feeding regimens of captive turtles are still limited. In the current study, the optimal dietary protein requirement for green turtle (Chelonia mydas) was investigated to support rearing in head-starting programs. Twenty-five-day-old turtles (44.5–46.2 g body weight, n = 45) were randomly distributed into 15 experimental plastic tanks, comprising three treatment replications of 3 turtles each. They were fed fishmeal-based feeds containing different levels of protein (30%, 35%, 40%, 45%, and 50%) for 8 weeks. At the end of feeding trial, growth performance (specific growth rate = 1.86% body weight/day) and feed utilization (protein efficiency ratio = 3.30 g gain/g protein) were highest in turtles fed with 40% protein in feed (p < .05). These nutritional responses were significantly supported by specific activities of fecal digestive enzymes, especially trypsin, chymotrypsin, amylase, and the amylase/trypsin ratio. Also, this dietary level improved the deposition of calcium and phosphorus in carapace, supporting a hard carapace and strong healthy bones. There were no negative effects in general health status of reared turtles, as indicated by hematological parameters. Based on a broken-line analysis between dietary protein levels and specific growth rate, the optimal protein level for green turtles was estimated as 40.6%. Findings from the current study support the use of artificial diets of specific protein levels to rear captive green turtle before release to natural habitats.     

Molecular Analysis of Echinostome Metacercariae from Their Second Intermediate Host Found in a Localised Geographic Region Reveals Genetic Heterogeneity and Possible Cryptic Speciation

Echinostome metacercariae are the infective stage for humans and animals. The identification of echinostomes has been based until recently on morphology but molecular techniques using sequences of ribosomal RNA and mitochondrial DNA have indicated major clades within the group. In this study we have used the ITS2 region of ribosomal RNA and the ND1 region of mitochondrial DNA to identify metacercariae from snails collected from eight well-separated sites from an area of 4000 km² in Lamphun Province, Thailand. The derived sequences have been compared to those collected from elsewhere and have been deposited in the nucleotide databases. There were two aims of this study; firstly, to determine the species of echinostome present in an endemic area, and secondly, to assess the intra-specific genetic diversity, as this may be informative with regard to the potential for the development of anthelmintic resistance and with regard to the spread of infection by the definitive hosts. Our results indicate that the most prevalent species are most closely related to E. revolutum, E. trivolvis, E. robustum, E. malayanum and Euparyphium albuferensis. Some sites harbour several species and within a site there could be considerable intra-species genetic diversity. There is no significant geographical structuring within this area. Although the molecular techniques used in this study allowed the assignment of the samples to clades within defined species, however, within these groupings there were significant differences indicating that cryptic speciation may have occurred. The degree of genetic diversity present would suggest the use of targeted regimes designed to minimise the selection of anthelmintic resistance. The apparent lack of geographic structuring is consistent with the transmission of the parasites by the avian hosts.     

Effects of ovarian endometriotic fluid exposure on fertilization rate of mouse oocytes and subsequent embryo development

Background

Accidental exposure of oocyte/cumulus complex to endometriotic fluid is not uncommon during oocyte retrieval. Only two studies were available on this subject and they gave conflicting results. In this study, we used a mouse model to evaluate the effect of controlled exposure of oocytes to ovarian endometriotic fluid.

Methods

Mouse oocytes/cumulus complexes (n?=?862) were divided into 4 groups, and were exposed to endometriotic fluid (group 1), pooled sera from subjects without endometrioma (group 2), phosphate-buffered saline (group 3), and fertilization medium (controls). After five minutes, oocytes were washed and inseminated. Embryo development was observed daily. The quality of hatching blastocysts was assessed by counting the number of inner cell mass (ICM) and trophectoderm (TE) cells.

Results

The fertilization, cleavage and blastocyst formation rates in the four groups were not statistically different. The proportions of hatching/hatched blastocysts from fertilized oocytes in groups 1 and 2 were significantly lower than those in group 3 and controls (P?=?0.015). Hatching blastocysts from all groups showed no significant difference in the number of ICM and TE cells.

Conclusions

Exposure of mouse oocytes/cumulus complexes to endometriotic fluid had subtle detrimental effects on subsequent blastocyst development. However, one should be cautious in projecting the results of this study to contaminated human oocytes in a clinical setting.     

Production of high activity Aspergillus niger BCC4525 β-mannanase in Pichia pastoris and its application for mannooligosaccharides production from biomass hydrolysis

A cDNA encoding β-mannanase was cloned from Aspergillus niger BCC4525 and expressed in Pichia pastoris KM71. The secreted enzyme hydrolyzed locust bean gum substrate with very high activity (1625 U/mL) and a relatively high k_cat/K_m (461 mg^?1 s^?1 mL). The enzyme is thermophilic and thermostable with an optimal temperature of 70 °C and 40% retention of endo-β-1,4-mannanase activity after preincubation at 70 °C. In addition, the enzyme exhibited broad pH stability with an optimal pH of 5.5. The recombinant enzyme hydrolyzes low-cost biomass, including palm kernel meal (PKM) and copra meal, to produce mannooligosaccharides, which is used as prebiotics to promote the growth of beneficial microflora in animals. An in vitro digestibility test simulating the gastrointestinal tract system of broilers suggested that the recombinant β-mannanase could effectively liberate reducing sugars from PKM-containing diet. These characteristics render this enzyme suitable for utilization as a feed additive to improve animal performance.     

Phosphate sensing in <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803: SphU and the SphS–SphR two-component regulatory system

The Pho regulon is controlled by the histidine kinase-response regulator pair SphS–SphR in many cyanobacteria and up-regulation of the Pho regulon can be monitored by measuring alkaline phosphatase activity. However, the mechanism regulating signal transduction between SphS and SphR has not been described. We have created a cyanobacterial strain allowing the introduction of mutations into the transmitter domain of SphS. Mutations at Thr-167, adjacent to the H motif of SphS, introduce elevated alkaline phosphatase activity in the presence of phosphate and an enhancement of alkaline phosphatase activity, when compared to the control strain, in phosphate-limiting media. SphU acts as a negative regulator of the SphS–SphR system in Synechocystis sp. PCC 6803 and we show that constitutive alkaline phosphatase activity in the absence of SphU requires signal transduction through SphS and SphR. However, constitutive activity in the absence of SphU is severely attenuated in the ΔSphU:SphS-T167N mutant. Our data suggest that Thr-167 contributes to the mechanism underlying regulation by SphU. We have also assembled a deletion mutant system allowing the introduction of mutations into SphR and show that Gly-225 and Trp-236, which are both conserved in SphR from cyanobacteria, are essential for activation of the Pho regulon under phosphate-limiting conditions.     

Frequency of pyrethroid resistance in Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in Thailand

Thirty‐two Aedes aegypti populations collected throughout Thailand and five populations of Aedes albopictus from southern Thailand were subjected to standard WHO contact bioassays to assess susceptibility to three commonly used synthetic pyrethroids: permethrin, deltamethrin, and lambda‐cyhalothrin. A wide degree of physiological response to permethrin was detected in Ae. aegypti, ranging from 56.5% survival (Lampang, northern Thailand) to only 4% (Kalasin in northeastern and Phuket in southern Thailand). All 32 populations of Ae. aegypti were found to have evidence of incipient resistance (62.5%) or levels of survival deemed resistant (37.5%) to permethrin. Four populations of Ae. albopictus were found with incipient resistance (97 – 80% mortality) and one with resistance (< 80%) to permethrin. The majority of Ae. aegypti populations (68.7%) was susceptible (> 98% mortality) to deltamethrin, with incipient resistance (observed 97–82% mortality) in other localities. In contrast, all populations of Ae. aegypti were completely susceptible (100% mortality) to the recommended operational dosage of lambda‐cyhalothrin. All five populations of Ae. albopictus were found completely susceptible to both deltamethrin and lambda‐cyhalothrin. Evidence of defined incipient or resistance to synthetic pyrethroids mandates appropriate response and countermeasures to mitigate further development and spread of resistance. In light of these findings, we conclude that routine and comprehensive susceptibility monitoring of dengue mosquito vectors to synthetic pyrethroids should be a required component of resistance management policies and disease control activities.     

First somatic mutation of E2F1 in a critical DNA binding residue discovered in well-differentiated papillary mesothelioma of the peritoneum

Background

Well differentiated papillary mesothelioma of the peritoneum (WDPMP) is a rare variant of epithelial mesothelioma of low malignancy potential, usually found in women with no history of asbestos exposure. In this study, we perform the first exome sequencing of WDPMP.

Results

WDPMP exome sequencing reveals the first somatic mutation of E2F1, R166H, to be identified in human cancer. The location is in the evolutionarily conserved DNA binding domain and computationally predicted to be mutated in the critical contact point between E2F1 and its DNA target. We show that the R166H mutation abrogates E2F1's DNA binding ability and is associated with reduced activation of E2F1 downstream target genes. Mutant E2F1 proteins are also observed in higher quantities when compared with wild-type E2F1 protein levels and the mutant protein's resistance to degradation was found to be the cause of its accumulation within mutant over-expressing cells. Cells over-expressing wild-type E2F1 show decreased proliferation compared to mutant over-expressing cells, but cell proliferation rates of mutant over-expressing cells were comparable to cells over-expressing the empty vector.

Conclusions

The R166H mutation in E2F1 is shown to have a deleterious effect on its DNA binding ability as well as increasing its stability and subsequent accumulation in R166H mutant cells. Based on the results, two compatible theories can be formed: R166H mutation appears to allow for protein over-expression while minimizing the apoptotic consequence and the R166H mutation may behave similarly to SV40 large T antigen, inhibiting tumor suppressive functions of retinoblastoma protein 1.